Recombinant cells that express phytate degrading enzymes in desired ratios

ABSTRACT

The present invention provides a recombinant combination strain which is capable of over-expressing at least two different genes under two separate promoters in filamentous fungi. The genes encode phytase and pH 2.5 acid phosphatase. Mixtures containing desired ratios of the two enzymes are prepared by recombinant DNA techniques. The enzyme mixtures show a cooperative effect in the degradation of phytic acid and its salts. The preferred ratios of the two enzymes are from about 3:1 to about 16:1.

This application is the U.S. national stage application of Internationalapplication Ser. No. PCT/US93/07058, filed Jul. 27, 1993, published asWO94/03072 Feb. 17, 1994, which was a continuation-in-part of U.S.application Ser. No. 07/925,401, filed Jul. 31, 1992, now abandoned, andclaims the benefit of the filing dates thereof under 35 U.S.C. § 120.

FIELD OF THE INVENTION

This invention is related to strains of filamentous fungi capable ofover-expressing at least two phytate degrading enzymes in desiredratios. Also disclosed are DNA sequences, promoters, DNA constructs andvectors useful for the preparation of such strains.

BACKGROUND OF THE INVENTION

Minerals are essential elements for the growth of all organisms. Forlivestock production of monogastric animals (e.g., pigs, poultry) andfish, feed is commonly supplemented with minerals. Plant seeds are arich source of minerals since they contain ions that are complexed withthe phosphate groups of phytic acid. Ruminants do not require inorganicphosphate and minerals because microorganisms in the rumen produceenzymes that catalyze conversion of phytate (myo-inositol-hexaphosphate)to inositol and inorganic phosphate. In the process minerals that havebeen complexed with phytate are released.

Phytate occurs as a source of stored phosphorus in virtually all plantfeeds (for a review see: Phytic Acid, Chemistry and Applications, E.Graf (Ed.), Pilatus Press: Minneapolis, Minn., U.S.A., 1986). Phyticacid forms a normal part of the seed in cereals and legumes. Itfunctions to bind dietary minerals that are essential to the new plantas it emerges from the seed. When the phosphate groups of phytic acidare removed by the seed enzyme phytase, the ability to bind metal ionsis lost and the minerals become available to the plant. In livestockfeed grains, the trace minerals bound by phytic acid are only partiallyavailable for absorption by monogastric animals, which lack phytaseactivity. Although some hydrolysis of phytate occurs in the colon, mostphytate passes through the gastrointestinal tract of monogastric animalsand is excreted in the manure contributing to fecal phosphate pollutionproblems in areas of intense livestock production. Inorganic phosphorusreleased in the colon has no nutritional value to livestock becauseinorganic phosphorus is absorbed only in the small intestine. Thus, asignificant amount of the nutritionally important dietary minerals arepotentially not available to monogastric animals.

Conversion of phytate to inositol and inorganic phosphorus can becatalyzed by microbial enzymes referred to broadly as phytases. Phytasessuch as the phytase #EC 3.1.3.8 are capable of catalyzing hydrolysis ofmyo-Inositol hexaphosphate to D-myo-inositol 1,2,4,5,6-pentaphosphateand orthophosphate. Certain fungal phytases reportedly hydrolyzeinositol pentaphosphate to tetra-, tri-, and lower phosphates; e.g., A.ficuum phytases reportedly produce mixtures of myoinositol di- andmono-phosphate (Ullah, 1988). Phytase producing microorganisms comprisebacteria such as Bacillus subtilis (V. K. Powar and V. J. Jagannathan,J. Bacteriol. 151:1102-1108, 1982) and Pseudomonas (D. J. Cosgrove,Austral. J. Biol. Sci. 23:1207-1220, 1970); yeasts such as Saccharomycescerevisiae (N. R. Nayini and P. Markakis, Lebensmittel Wissenschaft undTechnologie 17:24-26, 1984); and fungi such as Aspergillus terreus (K.Yamada, Y. Minoda and S. Yamamoto, Agric. Biol. Chem. 32:1275-1282,1968). The possible use of microbes capable of producing phytase as afeed additive for monogastric animals has been reported previously(Shieh and Ware, U.S. Pat. No. 3,297,548; Nelson, T. S. et al., J.Nutrition 101:1289-1294, 1971). To date, however, commercial applicationof this concept has not proved feasible, because of the high cost forproduction of microbial phytases.

Microbial phytases may also reportedly be useful for producing animalfeed from certain industrial processes, e.g., wheat and corn wasteproducts. The wet milling process of corn produces glutens sold asanimal feeds. Addition of phytase may reportedly improve the nutritionalvalue of the feed product. Fungal phytase enzymes and process conditions(t˜50° C. and pH˜5.5) have been reported previously in European PatentApplication 0 321 004. In processing soybean meal the presence ofphytate reportedly renders the meal and wastes unsuitable for feeds usedin rearing fish, poultry and other non-ruminants as well as calves fedon milk. Phytase is reportedly useful for improving the nutrient andcommercial value of this high protein soy material (see Finase EnzymesBy Alko, a product information brochure published by Alko Ltd.,Rajamaki, Finland). A combination of phytase and a pH2.5 optimum acidphosphatase from A. niger has been used by Alko, Ltd. as an animal feedsupplement in their phytic acid degradative product Finase F and FinaseS. A cost-effective source of phytase would greatly enhance the value ofsoybean meal as an animal feed (Shieh et al., 1969).

Phytase and less specific acid phosphatases are produced by the fungusAspergillus ficuum as extracellular enzymes (Shieh et al., 1969). Ullahreportedly purified a phytase from wild-type A. ficuum that had anapparent molecular weight of 61.7 kDa (on SDS-PAGE; as corrected forglycosylation); pH optima at pH2.5 and pH5.5; a Km of about 40 μM; and,a specific activity of about 50 U/mg (Ullah, A., Preparative Biochem.18:443-458, 1988); PCT patent application WO 91/05053 also reportedlydiscloses isolation and molecular cloning of a phytase from Aspergillusficuum with pH optima at pH2.5 and pH5.5, a Km of about 250 μM, andspecific activity of about 100 U/mg protein.

Acid phosphatases are enzymes that catalytically hydrolyze a widevariety of phosphate esters and usually exhibit pH optima below 6.0(Hollander, 1971); e.g., #EC 3.1.3.2 catalyzes hydrolysis oforthophosphoric monoesters to orthophosphate products. An acidphosphatase has reportedly been purified from A. ficuum. Thedeglycosylated form of the acid phosphatase has an apparent molecularweight of 32.6 kDa (Ullah et al., 1987).

Objects of the invention provide recombinant phosphatases isolated fromfilamentous fungi that improve the efficiency of release of phosphorusfrom phytate and the salts of phytic acid. Other objects of theinvention provide efficient and inexpensive sources of two or morerecombinant enzymes that are suitable for commercial use in feeds andindustrial processes with minimal processing.

SUMMARY OF THE INVENTION

Two enzymes were substantially purified from Aspergillus niger var.awamori strain ALKO243: namely, a phytase and a pH2.5 acid phosphatase.The amino acid sequence for isolated internal peptides was determinedfor each enzyme and the deduced nucleotide sequences were used toconstruct probes that were used to molecularly clone the two genes. Thegenomic nucleotide sequence was determined for each gene and the codingregion sequence was also determined (where necessary) by cloning thecDNA. Comparison of the deduced amino acid sequences of the phytase andpH2.5 acid phosphatase with other known phosphatases identified apotential enzyme active site sequence. Transformation vectors wereconstructed using promoters native to filamentous fungi and severaldifferent heterologous promoter sequences were compared in their abilityto drive expression of the respective genes. Recombinant host cells wereselected that over-produced phytase and pH2.5 acid phosphatase at levelsthat were about 2-fold to about 4000-fold higher, and 10-fold to126-fold higher (respectively), than the levels of these enzymessecreted by the ALKO243 (ATCC#38854) strain of Aspergillus.Dual-gene-transformed host cells were also selected that expressedelevated levels of the two recombinant enzymes, i.e., both phytase andpH2.5 acid phosphatase. Selected strains of dual-gene-transformed cellswere identified that synthesized and secreted phytase along with pH2.5acid phosphatase within desired tailor-made ranges of the respectiveenzyme activities, e.g., within a range of 3:1 to 16:1 pH2.5 acidphosphatase activity to phytase activity. The transformed recombinanthost cells disclosed herein solve the problems in the prior art andprovide cost-effective sources of commercial phosphatase enzymessuitable for use in commercial processes that liberate minerals fromphytates in plant materials either in vitro, i.e., in feed treatmentprocesses, or in vivo, i.e., by administering one or more of the enzymesto animals.

The phytase purified from A. niger var. awamori strain ALKO243(ATCC#38854; also known as IFO4033) exhibited an apparent molecularweight of 80-86,000 daltons (SDS-PAGE), and 45,000-48,000 daltons(SDS-PAGE), following treatment with endoglycosidase F/N-glycosidase F.The purified phytase appears to be monomeric under nondenaturingconditions with an isoelectric point of approximately 5.3. This phytaseenzyme exhibits activity in the absence of metal ions, i.e., metal ionindependent; the enzyme has a pH optima of about 5.0; and, a temperatureoptimum in the range of 55°-60° C.

The purified pH2.5 acid phosphatase from A. niger var. awamori strainALKO243 had an apparent molecular weight of about 66,000 daltons(SDS-PAGE) and 46,000-49,000 daltons (SDS-PAGE) after removal ofcarbohydrates with endoglycosidase F/N-glycosidase F. This purifiedpH2.5 acid phosphatase appears to be tetrameric under nondenaturingconditions with an isoelectric point of approximately 4 to 4.25; anapparent Km of 0.7 mM for sodium phytate at pH 2.5 and 4 mM forparanitrophenylphosphate; a pH optima of about pH2.5; and, temperatureoptimum of about 55° C.

Translated nucleotide sequences for phytase and pH2.5 acid phosphataseyielded polypeptides of 470 amino acids and 479 amino acids,respectively. The calculated molecular weights for the predicted phytaseand pH2.5 acid phosphatase polypeptides were approximately 51,400daltons and approximately 52,700 daltons, respectively.

The desired ratio of recombinant pH2.5 acid phosphatase phytase andphytase produced by a dual-gene-transformed recombinant host cellsachieves a balanced enzyme mixture in which cooperative enzyme activityrapidly and effectively catalyzes the near complete hydrolysis ofphytate to inositol and free phosphate with release of minerals from thephytic acid complex.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the pH optima of a metal ion independent phytase enzymepurified from Aspergillus niger var. awamori strain ALKO243 (ATCC#38854)with a pH optima of about pH5; and, >20% maximal enzyme activity in therange of pH2 to pH7 (as described in Example 1, below);

FIG. 2 shows the temperature optima at 55°-60° C., with >20% of maximalenzyme activity in the range of 25° C. to 65° C., for the purifiedphytase enzyme of FIG. 1, above,

FIG. 3 shows the pH optima at 37° C. of pH2.5 acid phosphatase purifiedfrom Aspergillus niger var. awamori strain ALKO243 that has an apparentKm of 0.7 mM for sodium phytate at pH2.5, a pH optima of pH2.0-2.5,and >20% activity in the range of pH1.5 to pH3.5 (as described below inExample 1);

FIG. 4 shows the temperature optima at about 55° C., and >20% maximalenzyme activity in the range of 25° C. to 60° C., for the pH2.5 acidphosphatase of FIG. 3, above,

FIG. 5 shows the relative phytase activity (i.e., liberation ofphosphate from sodium phytate) of Finase, a commercial preparationcontaining phytase and phosphatases, as a function of pH at 37° C.(squares) and 55° C. (+);

FIG. 6 shows the relative phytase activity (i.e., phosphate-liberatingactivity) of Finase (FIG. 5) as a function of temperature, i.e., between10° C. and 70° C., at pH5;

FIG. 7 shows an autoradiogram of radiolabeled degenerate oligonucleotidephytase probe PHY-1 hybridizing under stringent conditions withendonuclease fragments of genomic DNA generated with BamHI; EcoRI; XbaI;BamHI+EcoRI; BamHI+XbaI; and EcoRI+XbaI (as described in Example 2,below);

FIG. 8 shows an autoradiogram of radiolabeled specific oligonucleotidepH2.5 acid phosphatase probe PHY-31 hybridizing under stringentconditions with endonuclease fragments of genomic DNA generated withBamHI, HindIII, KpnI, PstI, SalI, SphI, or SstI (as described in Example2, below);

FIG. 9A and FIG. 9B shows the genomic nucleotide sequence (SEQ. ID.NO. 1) for the phytase gene in Aspergillus niger var. awamori strainALKO243 (ATCC#38854); the deduced amino acid sequence (SEQ. ID. NO. 2)for the phytase polypeptide, as described in Example 2, below; and, the5' regulatory promoter region of the gene;

FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D show the organization ofphytase expression vector constructs pFF1, pFF2, pFF3 and pFF4 (asdescribed in Example 4, below);

FIG. 11A and FIG. 11B shows the nucleotide sequence for the pH2.5 acidphosphatase gene (SEQ. ID. NO. 3) and the deduced amino acid sequence(SEQ. ID. NO. 4) of the enzyme;

FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D shows the organization ofphytase expression vector constructs pFF-6A, pFF8, pFF9, and pFF11 thatwere designed to remove any E. coli nucleotide sequences (as describedin Example 4, below);

FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D shows the organization ofpH2.5 acid phosphatase vector constructs pPHO-1-4A from which linearfragments can be isolated as described in Example 4, below;

FIG. 14A and FIG. 14B shows the organization of two transformationvectors having both the pH2.5 acid phosphatase gene and phytase gene,namely, pFIN-1A and pFIN-1B (as described in Example 4, below);

FIG. 15 shows phytase activity in a plate assay of transformed A. nigervar. awamori subclones that over-produce phytase by up to 1260-fold overthe levels produced by the parental ALKO243 strain. (The size of thecircles formed by the molybdate indicator complex around clones isproportional to enzyme activity; as described in Example 4, below;

FIG. 16 shows chromosomal phytase gene copy number and MRNA levels inphytase gene transformed A. niger var. awamori strain ALKO2268 asdetermined by Southern blot analysis and Northern analysis, respectively(as described in Example 4, below). The significant increase in phytaseactivity seen in FIG. 15 in transformed cells is attributable tointegration of one or more copies of the cloned recombinant phytase geneconstruct into chromosomal DNA (see Example 4, below);

FIG. 16A shows Southern blot analysis of untransformed control ALKO2268(lanes 1, 4 and 7) and over-producing phytase transformants (lanes 2, 5,8, 9 and 10);

FIG. 16B shows Northern blot analysis of mRNA levels in untransformedControl ALKO2268 (lane 1) and transformed strains (lanes 2-6);

FIG. 17 and FIG. 18 show chromosomal phytase and pH2.5 acid phosphatasegene copy numbers and mRNA levels in dual-gene-transformed A. niger var.awamori strain ALKO243 as determined by Southern blot analysis and byNorthern analysis, respectively (as described in Example 5);

FIG. 17A shows Southern blot analysis of untransformed control ALKO243(lane 2) and dual-transformed strains (lanes 3, 4 and 5) probed forphytase genes;

FIG. 17B shows Northern blot analysis of untransformed control (lane 1)and dual-transformed strains (lanes 2, 3 and 4) probed for phytase mRNA;

FIG. 18A shows Southern blot analysis of untransformed control ALKO243(lane 3) and dual-transformed strains (lanes 4, 5 and 6) probed forpH2.5 acid phosphatase genes;

FIG. 18B shows Northern blot analysis of untransformed control ALKO243(lane 1) and dual-transformed strains (lanes 2, 3 and 4) probed forpH2.5 acid phosphatase mRNA;

FIG. 19 graphically depicts the levels of phytase activity intwenty-four different transformants using circular or linear plasmidvector constructs (as described in Example 6, below); and

FIG. 20 shows the effects of different promoters on the levels ofrecombinant phytase activity and pH2.5 acid phosphatase activity indual-gene-transformed A. niger var. awamori strains ALKO243 andALKO2268, as described in Example 6, below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein the following terms are intended to mean as follows:

The term "phosphatase" is intended to mean an enzyme capable ofreleasing phosphate from a phosphate-containing substrate, e.g.,phytate. Representative examples of phosphatases include fungal phytasesand acid- and neutral-phosphatases such as pH2.5 acid phosphatase andpH6 neutral phosphatase.

The term "nucleic acid" is used herein to refer to natural or syntheticDNA and RNA, polynucleotides (i.e., greater than three nucleotides), andoligonucleotides (i.e., greater than nine nucleotides).

The term "capable of hybridizing under stringent conditions" is usedherein to mean annealing to a subject nucleotide sequence, or itscomplementary strand, under standard conditions, e.g., high temperatureand/or low salt content which tends to disfavor annealing of unrelatedsequences. A suitable protocol (involving 0.1×SSC and annealing at 68°C. for 2 hours) is described in Maniatis, T. et al., Molecular Cloning:A Laboratory Manual, Cold Springs Harbor Laboratory, Cold SpringsHarbor, N.Y., pp. 387-389, 1982.

The term "nucleotide sequence" is used herein to refer to a sequence ofnucleotides or nucleosides and may include non-contiguous sequences;e.g., in genomic sequences exon coding region sequences may beinterrupted by intron sequences and the like.

The term "contiguous nucleotide sequence" is used herein to refer to asequence of nucleotides linked in a serial array, one following theother.

The term "coding region" refers to the nucleotide sequence within anucleic acid that when transcribed and translated will give rise to asubject amino acid sequence, e.g., exon regions in genomic DNA that whentranscribed into mRNA will direct translation of the subject amino acidsequence. The term "encoded" is used to mean an amino acid sequencecoded for in the triplet code of nucleotides by the coding regionnucleotide sequence.

The term "transformation vector" is used herein interchangeably with"vector construct" to refer to a recombinant construct (e.g., a plasmidDNA) containing a subject phytase, and/or pH2.5 acid phosphatasenucleotide sequence incorporated therein, and includes as one subclass"expression vectors". Representative examples of transformation vectorsinclude plasmid vectors of E. coli (e.g, pBR322, pUC18, pUC19 and thelike as well as vectors useful for transforming filamentous strains offungi (e.g., pLO-3, pFF-6, pPHO-1 and the like). The subject phytase orpH2.5 acid phosphatase nucleotide sequence may contain the regulatorysequence elements from the 5' region of the respective gene (i.e.,termed herein "native regulatory elements"); or, a heterologous promoter(i.e., from a strain, variety, species or genus other than A. niger var.awamori strain ALKO243 or from a different gene, e.g., GA or GAPDHpromoter nucleotide sequences) may be added to drive expression of thesubject nucleotide sequence. The subject transformation vector may alsocontain suitable restriction sites for receiving additional nucleotidesequences useful for promoting chromosomal integration of the vectorDNA. The subject transformation vectors are capable of introducingphosphatase nucleotide sequences into a host cell so that they mayintegrate effectively into the host cell DNA, and so that the product ofthe coding region of the nucleotide sequence is expressed by thetransformed host cell.

"Promoter" is used to mean a nucleotide sequence capable of promotingexpression of a downstream nucleotide sequence such as transcription andtranslational regulatory signal sequences and the like. Representativeexamples of promoters are provided below in Examples 3-5, e.g., GA,GAPHD, and native phytase or pH2.5 acid phosphatase promoter.

"Selectable marker" is used to mean a nucleotide sequence capable ofencoding a polypeptide that when expressed by transformed cell confersupon the cell the ability to be identified or selected from among theother cells in a population of cells. Illustrative examples includeantigenic markers, phenotypic markers (e.g., cell size, shape, buddingpatterns and the like), and drug resistance markers such as phleomycinresistance.

As used herein the term "transformed recombinant host cell" refers to acell having chromosomal DNA with an integrated transformation vectornucleotide sequence therein. Representative examples of transformedrecombinant host cells of the invention include bacterial and fungalcells capable of synthesizing and/or secreting a recombinant phytaseand/or one or more recombinant phosphatases, i.e., a phytase encoded bya nucleotide sequence capable of hybridizing under stringent conditionswith SEQ. ID. NO. 1; and, the recombinant pH2.5 acid phosphatase, i.e.,a phosphatase encoded by a nucleotide sequence capable of hybridizingwith SEQ. ID. NO. 3.

The term "phytase nucleotide sequence" as used herein refers to thenucleotide sequence located within SEQ. ID. NO. 1.

The term "pH2.5 acid phosphatase nucleotide sequence" as used hereinrefers to a nucleotide sequence capable of hybridizing under stringentconditions with a nucleotide sequence in SEQ. ID. NO. 3.

The term "phosphatase" as used herein encompasses all enzymes capable ofcleaving a phosphate ester bond in a substrate, and includes phytases,and acid and neutral phosphatases.

The term "phytase" is intended to mean an enzyme capable of hydrolyzinga phosphate ester bond in a phytate substrate, e.g.,inositolhexaphosphate, inositolpentaphosphate, inositoltetraphosphate,and inositoltriphosphate and salts thereof

The term "purified phytase" is intended to mean a purified enzyme of theinvention isolated and substantially purified from A. niger var. awamoristrain ALKO243. The subject enzymes are monomeric under nondenaturingconditions with isoelectric points of about 5.3; apparent molecularweights on SDS-PAGE of about 80,000 daltons to 86,000 daltons, apparentmolecular weights after removal of carbohydrate (e.g., withendoglycosidase F/N-glycosidase F) of about 45,000-48,000 daltons(SDS-PAGE). The subject purified phytase enzymes have the followingcatalytic properties: namely, the enzymes are metal ion-independentenzymes capable of hydrolyzing a phosphate ester bond in a sodiumphytate substrate. The subject enzymes have a pH optima of about pH5 at37° C.; exhibit greater than 20% of maximal enzyme activity in the rangeof pH2 to pH7; and, have a temperature optima at 55° C. to 60° C.with >20% maximal enzyme activity in the range of 25° C. to 65° C. Oneunit of phytase activity (PU) is the amount of enzyme protein requiredto liberate 1 nmol of inorganic phosphate from sodium phytate in oneminute at 37° C. under the assay conditions described in Example 1,below.

The term "recombinant phytase" is intended to mean a phytase enzymeencoded by the nucleotide coding sequence of FIGS. 9A and 9B; SEQ. ID.NO. 1 and produced by a recombinant host cell transformed with thesubject transformation vector containing the subject phytase nucleotidesequence. The molecular weight predicted from transcription of thecoding region of subject phytase nucleotide sequence is a polypeptidetranslation product (i.e., prior to glycosylation) of about 51,000daltons to about 52,000 daltons.

The term "metal ion independent" is intended to mean that the activityof the enzyme can be measured in the absence of Mn²⁺ , Mg²⁺ and Ca²⁺.However, this is not intended to mean that the activity of the enzymemay not be increased in the presence Mn²⁺, Mg²⁺ or Ca²⁺.

The term "acid phosphatase" is used to mean an enzyme having a pHoptimum for mediating hydrolysis of a phosphate ester bond in asubstrate at a pH less than pH5.0, preferably less than pH3.0.

The term "pH2.5 acid phosphatase" is used to refer to a phosphatasehaving a pH optimum for mediating hydrolysis of a phosphate ester in asubstrate at pH2.0-2.5, e.g., a sodium phytate substrate.

The term "purified pH2.5 acid phosphatase" is intended to mean apurified enzyme of the invention isolated and substantially purifiedfrom A. niger var. awamori strain ALKO243. When purified from thesubject strain of filamentous fungi the enzyme has the followingproperties: namely, the enzyme isolated under nondenaturing conditionsis a glycoprotein tetrameric complex of four identical monomers with anapparent isoelectric point of about 4.0 to about 4.25. Each of themonomers has an apparent molecular weight of about 66,000 daltons underdenaturing conditions in SDS-PAGE, and the monomers have an apparentmolecular weight of 46,000-49,000 daltons (SDS-PAGE) after substantialremoval of carbohydrate. The subject purified pH2.5 acid phosphataseenzymes have the following catalytic properties: namely, the enzymes cancatalyze hydrolysis of a phosphate ester bond in a sodium phytatesubstrate with an apparent Km of about 0.7 mM (i.e., at 37° C. andpH2.5, as described in Example 1, below). The subject enzymes have a pHoptima of about pH2 to about pH2.5 (i.e., at 37° C.); have greater than20% of maximal enzyme activity in the range of pH1.5 to pH3.5; and, havea temperature optima at 55° C. with >20% maximal enzyme activity in therange of 25° C. to 60° C. One unit of pH2.5 acid phosphatase activity(HFU) is the amount of enzyme protein required to liberate 1 nmol ofinorganic phosphate from p-nitrophenyl phosphate in one minute at 37° C.under the conditions described in Example 1, below.

The term "recombinant pH2.5 acid phosphatase" is intended to mean apH2.5 acid phosphatase enzyme encoded by the coding region of anucleotide sequence capable of hybridizing under stringent conditionswith a nucleotide sequence in FIGS. 11A and 11B; (SEQ. ID. NO. 3 ) andproduced by a recombinant host cell transformed with the subjecttransformation vector containing the subject pH2.5 acid phosphatasenucleotide sequence. In a representative example, the subject pH2.5 acidphosphatase nucleotide sequence is the coding region of SEQ. ID. NO. 3that encodes a polypeptide with an apparent molecular weight (i.e.,prior to glycosylation) of about 52,000 daltons to about 53,000 daltons.

The term "secreted" is used herein to refer to the extracellular form ofa protein, e.g., a "secreted" protein that is synthesized in a cell andtransported into the extracellular culture medium where its presence orenzyme activity may be assayed.

The term "phytase enzyme activity" is used herein to refer to thecatalytic activity of a phytase, e.g., in mediating hydrolysis of asodium phytate substrate to release phosphate, as may be convenientlymeasured in an assay for phosphate such as that described in theExamples, below.

The term "pH2.5 acid phosphatase enzyme activity" is used herein torefer to the phosphatase activity of a pH2.5 acid phosphatase, e.g., inmediating hydrolysis of a phosphate ester bond in aparanitrophenylphosphate substrate (in an assay such as that describedin the Examples, below).

The term "over-producing" is used interchangeably with the term"over-expressing" to indicate that the subject transformed recombinanthost cell is capable of synthesizing and secreting levels of the subjectenzyme that are at least about 2-fold higher than the amount of enzymesynthesized under identical conditions by cells of A. niger var. awamoristrain ALKO243 (ATCC#38854). As illustrated in Example 4, (Tables 8 and9, below), over-production in strain ALKO2268 results in secretion inskake flask cultures conducted according to Example 1 of about 45-foldmore phytase enzyme activity per ml of the culture media than thatsecreted under equivalent conditions by ALKO243. Illustrativetransformants with the subject nucleic acids of the invention (i.e.,also shown in Example 4) produce about 6-fold greater activity thanALKO2268 (i.e., see Table 7; up to about 300-fold greater activity thanALKO243). Other transformants in Example 4 (i.e., Tables 8 and 9)produced phytase activities per ml up to about 2,100-fold greater thanALKO243. Over-production in respect to pH2.5 acid phosphatase issimilarly illustrated by transformants in Example 4, below, thatproduced up to about 130-fold (i.e., Table 11) greater activity per mlthan ALKO243. By comparison, strain ALKO2268 produces approximately41-46% of the pH2.5 acid phosphatase enzyme activity produced byALKO243.

The term "ratio of pH2.5 acid phosphatase to phytase" refers to theratio of a pH2.5 acid phosphatase enzyme activity to a phytase activity,in this case, a ratio that may be calculated by dividing the enzymeactivity of pH2.5 acid phosphatase per ml of sample (e.g., the number ofHFU/ml) by the phytase enzyme activity per ml of sample (e.g., thenumber of PU/ml). Representative assays for determining phytase andpH2.5 acid phosphatase activity are provided in the Examples, below. Forcomparative purposes, the results presented in the Examples below showthe amount of phytase and pH2.5 acid phosphatase activity, respectively,produced and secreted into the culture broth by A. niger var. awamoristrain ALKO243 (ATCC#38854) over 5 days of fermentation culture underthe conditions described in Tables 7 and 8, below (Example 3). Cells ofALKO243 cultured under these conditions produce about 80-450 PU ofphytase and about 5,000-6,000 HFU of pH2.5 acid phosphatase; and, forreference purposes, the over-producer phytase strain A. niger var.awamori strain ALKO2268 produces about 3,000-9,000 PU and 2,000-3000HFU. With these exemplary strains the ratio of pH2.5 acid phosphatase tophytase (i.e., HFU/PU) is about 0.6 in shake flask fermentation culturemedia obtained from ALKO2268 after 5 days, and is 64.7 with ALKO243,(e.g., see Tables 11 and 14-16, below). As illustrated in Example 5,below, ratios of pH2.5 acid phosphatase activity (HFU) to phytaseactivity (PU) ranged from about 4 to about 16 with transformants shownin Table 14, and about 3 to 6 with the transformants whose enzymesactivities are shown in Tables 15 (PU) and Table 16 (HFU). The subject"ratio of pH2.5 acid phosphatase to phytase" achieves a balanced enzymemixture in which cooperative enzyme activity rapidly and effectivelycatalyzes the near complete hydrolysis of phytate to inositol and freephosphate with release of minerals from the phytic acid complex at a pH(e.g., that in the stomach of a monogastric animal) and temperaturedesired in a commercial product. The term "cooperative enzyme activity"is used to mean that the subject ratios confer upon the mixture ofenzymes the properties of: a) more rapid catalysis of phytate toinositol and free phosphate; b) more efficient conversion of phytate toinositol and free phosphate; and, c) more complete conversion of phytate(i.e., IP6, see the Examples below) to inositol and phosphate (i.e.,greater than 80% conversion as illustrated in Example 1, Table 1,below).

One "phytase normalized unit" or "PNU" is defined as the amount ofphytase activity produced by the A. niger ALKO243, which in this case isequivalent to 85 PU/ml. One "acid phosphatase normalized unit" or "APNU"is defined as the amount of acid phosphatase activity produced by the A.niger ALKO243 strain, which in this case is equivalent to 5500 HFU/ml.

Embodiments of the invention provide phytase nucleotide sequencescapable of hybridizing under stringent conditions with a nucleotidesequence of SEQ. ID. NO 1. The invention also provides pH2.5 acidphosphate nucleotide sequences capable of hybridizing under strigentconditions with a nucleotide sequence of SEQ. ID. NOS. 6 or 9. Thesubject nucleotide sequences are useful for constructing oligonucleotideprobes, transformation vectors, transformed recombinant host cells,encoding recombinant phytase and pH2.5 acid phosphatase proteins, andthe like, as described below.

In other embodiments the invention provides transformed recombinant hostcells, e.g., E. coli, Bacillus, Aspergillus, Trichoderma, Penicillium,Cephalosporium, Rhizopus, and the like, that have copies of the subjectphytase and/or pH2.5 acid phosphatase nucleotide sequences of theinvention. In a preferred embodiment the transformed recombinant hostcells are selected from among species of filamentous fungi, e.g.,Aspergillus, Trichoderma, and Rhizopus; and, in another preferredembodiment the subject host cells are selected from among varieties andstrains of Aspergillus niger. In a most preferred embodiment the subjecthost cells are selected from among recombinant host cells having phytasenucleotide sequences, e.g., transformed cells of the phytaseover-producing strains GAI-6, GAL-142, GAN-1, GAG-12, GAO-248, GAI-12,GAK4-46, GAI-2, GAK4-52, GAM-111, GAK4-47, GAM-225, GAD-103, GAD-23,GAD-103, GAD-23, GAD-130, GAM-199, GAE-3, GAE-32, GAM-111, and GAL-65(as described in Example 4, below). In another preferredembodiment thesubject host cells are selected from among recombinant host cellstransformed with vectors having pH2.5 acid phosphatase nucleotidesequences, e.g., transformed cells of strains GAO-69, GAW-131, GBL-128,GBL-97, GAO-61, GAW-89, GAW-130, GAW-121, GBL-87, GBL-119, GAO-84,GAW-54, GBL-129, GAW-141, GBL-103, GAW-112, GBL-92, GAW-114, and GAT-143(as described in Example 2, 4, 5, or 6 below). Other preferred hostcells include cells selected from the transformed strains GAX-11,GAX-12, GBE-14, GBH-134, GBH-15, GBJ-9, GBJ-10, GBJ-13, GBJ-16, GBJ-26,GBJ-27, GBJ-28, GBJ-31, GBJ-35, GBJ-38, GBJ-40, GBJ-76, and GBJ-82 (asdescribed below in Example 2, 4, 5, or 6). Those skilled in the art willrecognize that the nucleotide sequences, transformation vectors andtransformed recombinant host cells provided herein are useful foridentifying additional strains having substantially the same propertiesas the afore-identified strains.

Skilled artisans will recognize that the nucleotide sequences anddeduced amino acid sequences of the invention may be useful inconstructing nucleotide and antibody probes for identifying andisolating natural variants, and mutants of transformants, e.g., mutantsresulting from treatment with chemicals, UV, gamma-irradiation and thelike). Mutants may have increased expression of the subject phytase,pH2.5 acid phosphatase, or both a phytase and a pH2.5 acid phosphatase.Representative screening assay for identifying the latter mutantsinclude Northern and Southern blotting, and Western blotting withantibodies directed to peptides (natural and synthetic) within thededuced amino acid sequences of the subject phytases and pH2.5 acidphosphatases.

Those skilled in the art will of course recognize that mixtures oftransformed recombinant host cells may be used to achieve production ofa phytase and one or more phosphatases; e.g., transformed recombinanthost cells from a strain producing phytase may be mixed with transformedrecombinant host cells from a strain producing pH2.5 acid phosphatase.In this manner, the mixed cell cultures may be constructed so that thecells release a desired ratio of phytase enzyme activity to pH2.5 acidphosphatase enzyme activity.

The subject transformed recombinant host cells of the invention may alsobe used for preparing substantially pure recombinant phytasepreparations. In a preferred embodiment phytase nucleotide sequences inthe transformed recombinant host cell encode a polypeptide having aphytase amino acid sequence RHGXRXP SEQ. ID. NO. 5, wherein R isarginine, H is histidine, G is glycine, X is any amino acid, and P isproline.

Embodiments of the invention also provide "mixtures" of recombinantphytase and pH2.5 acid phosphatase in varying states of purity andformulated in a desired ratio of substantially pure pH2.5 acidphosphatase to phytase enzyme activity. Formulating such a balancedmixture of enzymes in the desired ratios confers upon the mixture theproperty of cooperative enzyme activity (described above) that can betailor-made to encompass the range of properties desired in a selectedcommercial application (e.g., uses such as those described below).Starting material for the recombinant phytase and recombinant pH2.5 acidphosphate include fermentation broths, production culture media, and thelike from transformed recombinant host cells or from selected strainsthat over-produce the subject phytase and/or pH2.5 acid phosphatase.Down-stream processing of the subject enzymes into a product may involveremoval of cells and cellular debris (e.g., by centrifugation,filtration and the like), followed by concentration (e.g., byultrafiltration, ion exchange or affinity chromatography and the like),or the starting material may be suitable for use in commercial processesafter a simple purification (e.g., lyophilization). The subject mixturesmay be prepared by combining equal (or different) amounts of the subjectenzyme preparations (e.g., from different cell cultures), in order toachieve the desired ratio of the respective enzyme activities, or thesubject mixtures may be existent in the same culture (e.g., the productof a dual-gene-transformed recombinant host cell, as described below).In a preferred embodiment the subject mixture contains a ratio of aphosphatase enzyme activity (e.g., pH2.5 acid phosphatase) to a phytaseenzyme activity that is about 3:1 to about 16:1.

Embodiments of the invention provide transformed recombinant host cellsthat are constructed with one or more transformation vectors having aphytase nucleotide sequence and one or more phosphatases, i.e., a pH2.5acid phosphatase nucleotide sequence, wherein expression of each enzymeunder the control of an independent promoter sequence. The subjecttransformed host cells are selected for expression of the two (or more)protein products within a desired range of enzyme activities.

The phosphatases produced by the transformed recombinant host cells andprocesses that are embodiments of the invention provide the followingadvantages over other enzyme preparations used previously in the art:namely, the subject phosphatases of the invention have higher enzymeactivity per unit volume of sample, greater yield of enzyme protein perunit volume of sample, greater cost-efficiency, less concentrationand/or purification required for use in a commercial product or process,greater efficacy in converting phytate to free inositol and inorganicphosphate, and fewer digestive side-effects when used in animal feeds.

The subject phosphatases produced by the transformed recombinant hostcells of the invention are useful in commercial processes for releasingminerals from complexes with phytate in plant materials such as seedsand waste matter of milling, e.g., soybean meal, so that low valuematerials are converted efficiently and effectively to a high qualityfeed for non-ruminant animals. Examples of commercial processes in whichthe subject enzymes may be useful include corn wet milling, plantprotein isolation (especially soy protein isolation), cereal treatmentfor use in baking, and the like.

In a preferred embodiment the subject phosphatases of the invention areadded directly to animal feeds so that phosphates are ingested by theanimal and released in vivo in the digestive tract, e.g., of anon-ruminant animal. In this case the subject phosphatases are selectedto have pH optima for their respective enzyme activities that coincidewith the digestive pH encountered in a non-ruminant animal (i.e., therange of pH1 to pH6).

Suitable methods for preparing the subject enzymes for use in productsinclude spray drying, stabilization in liquid formulations, granulation,and encapsulation, which are known to those skilled in the art.

The subject phosphatase enzymes of the invention, and subject mixturesof enzymes, are capable of degrading phytate to free phosphate moreefficiently and rapidly than any one of the constituent enzymes alone.Embodiments of the invention provide a mixture of a phytase and a pH2.5acid phosphatase that is capable of degrading inositolphosphates ofphytates and phytic acids, inositolhexaphosphate, IP6;inositolpentaphosphate, IP5; inositol-tetraphosphate, IP4;inositoltriphosphate, IP3, inositol diphosphate, IP2; inositolmonophosphate, IP1; to free inositol and inorganic phosphate. Thesubject mixture provides cooperative enzyme activities by constructingan enzymatic cascade for more rapid, complete, and efficient conversionof a phytate substrate to inorganic phosphate and inositol. For example,a phytase having phytate (IP6) as a preferred substrate may catalyzeefficient hydrolysis of IP6 to IP5, IP4, IP3, and IP2 but not to freeinositol and inorganic phosphate. In turn, a pH2.5 acid phosphatase mayprefer simple phosphate substrates (e.g., IP5, IP4, IP3, IP2 and IP1)and may catalyze efficient hydrolysis of these substrates to freeinositol and inorganic phosphates. It is believed that by formulatingthe subject phosphatase of the invention within desired optimum rangesof phytase to pH2.5 acid phosphatase activity the subject mixtures ofthe invention provide balanced enzyme mixtures having cooperative enzymeactivity. The subject mixtures of the invention formulated in thismanner may provide more rapid, efficient and complete release of greateramounts of free inositol and inorganic phosphate from phytate and phyticacid than produced in the same time (and under the same conditions of pHand temperature) by either of the constituent phosphatase enzymes.

EXAMPLE 1 Purification And Amino Acid Sequencing Of Phosphatases PhytaseAnd Acid-Phosphatase Peptides

General materials and methods are described in the section entitled"Materials and Methods", which follows at the end of this and eachsubsequent Example.!

Purification of phytase and pH2.5 acid phosphatase:

Phytase and pH2.5 acid phosphatase enzymes were purified at 4 °-8° C.(unless otherwise stated) from the cell-free culture mediumfiltrate/concentrate of A. niger var. awamori strain ALKO243(ATCC#38854). The culture filtrate/concentrate (990 ml) was adjusted (onice) to 70% saturation in ammonium sulfate, and the precipitate wasremoved by centrifugation at 10,000×g for 15 min. The supernatant (1070ml) was next separated by hydrophobic chromatography on Octyl-SepharoseCL-4B (Pharmacia). The column (5 cm×17 cm) was equilibrated in a 20 mMbis-Tris/HCl buffer, pH6.2, containing 0.436 g (NH₄)₂ SO₄ per ml;supernant was applied; and, non-adsorbed proteins were removed bywashing with 500 ml of the equilibration buffer solution. Adsorbedproteins were eluted from the column using a 500 ml linear gradient from70% to 0.0% of ammonium sulfate in 20 mM bis-Tris/HCl buffer, pH6.2. Tenml fractions were collected and analyzed for phytase and pH2.5 acidphosphatase enzyme activity. Most phytase activity eluted early in thegradient. Fractions containing the respective different enzymeactivities were pooled separately; concentrated by ultrafiltration onAmicon PM10 membrane filters. Phytase containing fractions were desaltedby passage over PD10 (Pharmacia) gel filtration columns equilibrated in50 mM bis-Tris/HCl, pH6.2 buffer. Phytase purification is describedfirst, followed by acid phosphatase purification.

Phytase was purified first by anion exchange chromatography onDEAE-Sepharose (Pharmacia). Briefly, a 24.5 ml aliquot was applied to a5 cm×7 cm column equilibrated in 50 mM bis-Tris/HCl, pH6.2. Non-adsorbedproteins were removed by washing with the equilibration buffer (100 ml),and adsorbed proteins were eluted using a linear 200 ml gradient from0.0M to 0.5M NaCl, in equilibration buffer. The fractions were assayedfor phytase activity and fractions with activity were pooled andconcentrated to 600 μl using a Centricon-30 miniconcentrator. Portionsof 100 μl were applied at about 23° C. to a Superose 12 HR 10/30 HPLCcolumn (Pharmacia) and proteins were eluted with 50 mM bis-Tris/HCl,pH6.2 at a flow rate of 0.3 ml/min. The active fractions wereidentified, pooled, and concentrated and transferred into 50 mM sodiumformate buffer, pH3.8 using a Centricon-30 microconcentrator. The enzymesolution in formate buffer was purified further using cation exchangechromatography. Samples of enzyme were applied in 2 ml aliquots to aMono S HR 5/5 FPLC column (Pharmacia) equilibrated in 50 mM sodiumformate (pH3.8) at about 23° C. The column was washed with theequilibration buffer (10 ml) and the bound protein was eluted at 60ml/hr. using a 20 ml linear gradient from 0 mM to 430 mM NaCl in theformate equilibration buffer. Phytase was purified by this method with ayield of 18.4% that had a specific activity of approximately 275,900(PU/mg) with a calculated purification of 130-fold (Table A).

                  TABLE A                                                         ______________________________________                                        Summary of purification of phytase                                                        Total    Total   Specific    Purifi-                                          Activity Protein Activity                                                                            Yield cation                               Step        (PU)     (mg)    (PU/ml)                                                                             (%)   (fold)                               ______________________________________                                        Culture filtrate                                                                          4,486,680                                                                              2,119    2,117                                                                              100    1                                   Ammonium sulphate                                                                         3,771,750                                                                              1,263    2,986                                                                              84.1    1.4                                supernatant                                                                   Octyl Sepharose                                                                           1,765,881                                                                              32.3     54,671                                                                             39.4  26                                   DEAE-Sepharose                                                                            1,453,470                                                                              8.4     173,032                                                                             32.4  82                                   Superose 12 1,010,888                                                                              5.7     177,349                                                                             22.5  84                                   Mono S        827,566                                                                              3.0     275,885                                                                             18.4  130                                  ______________________________________                                    

Acid phosphatase containing fractions from the pooled Octyl-Sepharosefraction, above, were first concentrated by ultrafiltration on AmiconPMI 10 filter, and then subjected to molecular-sieve chromatography on a2.6 cm×94 cm Sephacryl S-200 (Pharmacia) column equilibrated in 50 mMbis-Tris/HCl (pH6.2). Proteins were eluted at 20 ml/hr and fractionswith activity were pooled and separated further by anion exchangechromatography on a 5 cm×7 cm DEAE-Sepharose (Pharmacia) columnequilibrated in 50 mM bis-Tris/HCl, pH6.2. The column was washed with100 ml of equilibration buffer and adsorbed proteins were eluted using a200 ml linear gradient from 0.0M to 0.5M NaCl in equilibration buffer.Pooled active fractions were then concentrated; transferred to 20 mMbis-Tris/HCl, pH6.0 by ultrafiltration on an Amicon PM10 membrane; and,subjected to a second step of anion exchange chromatography, this timeusing a Mono Q HR 5/5 HPLC column (Pharmacia) equilibrated in 20 mMbis-Tris/HCl, pH6.0 at about 23° C. The sample was applied in 3.5 mlaliquots; the column was washed with 10 ml of the equilibration buffer;and, the proteins were eluted at 60 ml/hr using a 20 ml linear gradientfrom 0.0 mM to 350 mM NaCl in the equilibration buffer. Fractionscontaining enzyme activity were pooled, concentrated to a total volumeof 400 μl, and transferred into 20 mM bis-Tris/HCl, pH6.2 containing 150mM NaCl using a Centricon-30 microconcentrator. Additional purificationwas accomplished, first by molecular-sieve chromatography on a Superose12 HR 10/30 HPLC column (Pharmacia) equilibrated in the bis-Tris/HClsample buffer. Aliquots of 100 μl were applied to this column andproteins were eluted at 23° C. at a rate of 18 ml/hr. Fractions containenzyme activity were pooled, transferred into 20 mM histidine/HCl, pH5.8buffer using a PD10 gel filtration column, and subjected to a secondstep of purification by anion exchange chromatography on a Mono Q HR 5/5HPLC column. Aliquots of 1 ml were applied to the column, the column waswashed with 5 ml of the histidine/HCl sample buffer at about 23° C.Proteins were eluted at a rate of 60 ml/hr using a linear 20 ml gradientfrom 0 mM to 350 mM NaCl in equilibration buffer. The pH2.5 acidphosphatase was purified by this method at 13% yield with a 126-foldpurification, Table B.

                                      TABLE B                                     __________________________________________________________________________    Summary of purification of pH 2.5 Phosphatase                                           Total  Total Specific  Purifi-                                                Activity                                                                             Protein                                                                             Activity                                                                            Yield                                                                             cation                                       Step      (HFU)  (mg)  (HFU/mg)                                                                            (%) (fold)                                       __________________________________________________________________________    Culture filtrate                                                                        116,523,000                                                                          2,119  54,990                                                                             100 1                                            Ammonium sulphate                                                                       88,275,000                                                                           1,263  69,893                                                                             75.8                                                                              1.3                                          supernatant                                                                   Octyl Sepharose                                                                         68,296,470                                                                             583 117,147                                                                             58.6                                                                              2.1                                          Sephacryl 52,237,600                                                                             97.9                                                                              533,581                                                                             44.8                                                                              9.7                                          DEAE-Sepharose                                                                          46,127,692                                                                             54.6                                                                              844,830                                                                             39.6                                                                              15.4                                         Mono Q    19,326,753                                                                               3.28                                                                            5,892,303                                                                           16.6                                                                              107                                          Superose  16,876,978                                                                           nd    nd    14.5                                                                              nd                                           Mono Q    15,197,050                                                                              2.2                                                                              6,907,750                                                                           13.0                                                                              126                                          __________________________________________________________________________     nd = not determined                                                      

Phytase: Phytase as purified by the methods recited (above) exhibited anapparent molecular weight on SDS-PAGE of approximately 80,000-86,000daltons. The protein assubstantially purified (i.e., on SDS-PAGE) gave apositive reaction with periodic acid Schiff staining for carbohydrate(i.e., the purified phytase enzyme is a glycoprotein). After treatingthe purified phytase with a mixture of endoglycosidase F/N-glycosidase Fthe apparent molecular weight of the enzyme (on SDS-PAGE) was shifted toabout 45,000-48,000 daltons, suggesting that about 44% of the molecularmass may be attributable to carbohydrate. (The type of carbohydratelinkage and the nature of the moiety was not determined.) The molecularweight of the undenatured purified phytase enzyme was shown to beapproximately 90,000 daltons by molecular-sieve gel filtration (i.e.,based on its Stokes radius), and an apparent molecular weight of 100,000daltons was observed by native gradient gel PAA electrophoresis. Thelatter results suggest that the undenatured purified phytase enzymeexists as a monomer. The isoelectric point of the subject phytase is 5.3by isoelectric focusing.

pH2.5 acid phosphatase: pH2.5 acid phosphatase, purified by the methodsrecited above, exhibited an apparent subunit molecular weight onSDS-PAGE of 66,000 daltons. The substantially purified enzyme (i.e., onSDS-PAGE) gave a positive reaction with periodic acid Schiff stainingindicating that pH2.5 acid phosphatase is also a glycoprotein. Afterremoving carbohydrate with endoglycosidase F/N-glycosidase F the proteinexhibited an apparent molecular weight of 46,000-49,000 daltons bySDS-PAGE. (The glycosidic linkage and nature of the carbohydrate moietywas not characterized.) pH2.5 acid phosphatase exhibits an apparentmolecular weight of 280,000 daltons by native gradient PAA gelelectrophoresis, suggesting that the purified undenatured enzyme existsas a tetramer of four 66,000-dalton subunits. The isoelectric point ofacid phosphatase by isoelectric focusing is approximately 4 to 4.25.

Catalytic properties of the purified phytase and pH2.5 acid phosphataseenzymes:

Phytase: The purified phytase exhibited optimal enzyme activity (asmeasured at 37° C., in conditions of excess substrate), at a pH ofapproximately pH5.0 with a shoulder at pH2.5 and >20% of maximal enzymeactivity in the range of pH2 to pH7 (FIG. 1). The temperature optimumfor the purified phytase enzyme (at optimal Na-phytate substrate andpH5.0) was found to be in the range of 55°-60° C. with >20% of maximalenzyme activity in the range of 25° C. to 65° C. (FIG. 2). The purifiedphytase enzyme catalyzed hydrolysis of sodium phytate in the absence ofadded metal ion (i.e., the enzyme exhibited no absolute requirement formetal ions and thus was "metal ion independent"); however, the activityof the purified phytase enzyme was increased in the presence of Mn²⁺,Mg²⁺ and Ca²⁺.

pH2.5 acid phosphatase: The apparent Km of the pH2.5 acid phosphatasefor Na-phytate substrate at 37° C. and pH2.5 was determined to be about0.7 mM; and, for paranitrophenylphosphate (PNP) at 37° C. and pH2.5 theapparent Km was 4 mM. The purified pH2.5 acid phosphatase enzymeexhibited a pH optimum in the range of pH2.0 to pH2.5 (i.e., at 37° C.,in the presence of optimal concentrations of Na-phytate substrate)with >20% of maximal activity in the range of pH1.5 to pH3.5 (FIG. 3).The temperature optimum for pH2.5 acid phosphatase-mediated hydrolysisof Na-phytate was found to be about 55° C. with >20% of maximal enzymeactivity in the range of 25° C. to 60° C. (FIG. 4). For comparison thepH/activity profile of a commercial non-purified Finase preparation isshown in FIG. 5, and the temperature/activity profile is shown in FIG.6.

Degradation of phytate by phytase and pH2.5 acid phosphatase andmixtures thereof: Finase is a commercial preparation of enzymes fromAspergillus that includes a mixture of phytase and phosphatase. Theratio of pH2.5 acid phosphatase (HFU) activity to phytase activity (PU)of Finase was determined to be approximately 7:1. A comparison was madeof the rate of phytate degradation by a commercial Finase enzymepreparation with the rate of the degradation by preparations of purifiedphytase, purified pH2.5 acid phosphatase and by mixtures thereofcontaining different acid phosphatase to phytase ratios. The ratio ofacid phosphatase activity (HFU) to phytase activity (PU) of the purifiedphytase is approximately 0.4:1 whereas the acid phosphatase exhibits nophytase activity at pH5.0. The ratios of the enzyme mixtures areindicated in Tables 1.a and 1.b. The comparison was made at pH2.5 37° C.and at pH5.0 37° C. using 10 mM Na-phytate as substrate. The enzymeactivities of all different preparations used in the experiments atpH2.5 were 10,000 HPU per mmol of Na-phytate substrate (HPU is the unitof phytase activity measured at pH2.5 using 0.2M glycine (HCl) bufferpH2.5 instead of Na-citrate buffer, see Materials and Methods at the endof Example 1). In the experiments conducted at pH5.0 the enzyme dosageper mmol of Na-phytate substrate was 10,000 PU, with the exception ofthe experiment carried out using purified acid phosphatase alone.Samples were taken from the reaction mixture after the times indicated(hours, h; 0, 1, 2, 8, 24, and 48h); the samples were freeze-dried andthen later analyzed for inositol hexa-, penta-, tetra- and tri-phosphates (i.e., IP6, IP5, IP4 and IP3, respectively), as well as forfree inositol (Ins) and inorganic phosphorus (Pi). Inositol hexa-,penta-, tetra-, and triphosphates were analyzed by the method ofSandberg et al. (1987). Inositol was analyzed by HPLC using Sugar Pak 1column (300 mm×6.5 mm, Waters), 0.1 mM Ca-EDTA as eluent(Calcium-Titriplex Merck 8439), flow 0.6 ml/min at 90° C. The detectionwas by RI (Waters), using myo-inositol (Fluka) 0.1-0.5 mg/ml as thestandard. Inorganic phosphorus was analyzed as described in Materialsand Methods in the end of Example 1. The results of these experimentsare presented in Table 1a and Table 1b, below.

                                      TABLE 1a                                    __________________________________________________________________________    pH 2.5          Yield, % of Theoretical.sup.a                                 Enzyme(s)       Time                                                          dosage/mmol substrate                                                                         (h)                                                                              IP6                                                                              IP5                                                                              IP4                                                                              IP3                                                                              Ins                                                                              P.sub.i                                     __________________________________________________________________________                    0  100                                                                              0  0  0  0                                              Finase          1  40 13 19 0  0                                              10,000 HPU      2  39 20 51 8  0                                                              8  0  0  2  38 5                                                              24 0  0  0  0  57                                                             48 0  0  0  0  94 103                                                         0  100                                                                              0  0  0  0                                              Phytase         1  20 29 20 0  0                                              10,000 HPU      2  5  30 68 22 0                                                              8  0  0  (+)                                                                              24 0                                                              24 0  0  0  0  0                                                              48 0  0  0  0  0  87                                                          0  100                                                                              0  0  0  0                                              pH 2.5 Acid Phosphatase                                                                       1  67 8  26 0  0                                              10,000 HPU      2  54 . 9                                                                              37 (+)                                                                              0                                                              8  26 2  42 12 1                                                              24 2  1  8  4  40                                                             48 1  0  5  67 88                                                             0  100                                                                              0  0  0  0  0                                           Phytase 9,000 HPU                                                                             1  44 23 32 20 0  16                                          pH 2.5 Acid Phosphatase 1,000 HPU                                                             2  6  13 71 17 0  31                                                          8  0  2  10 0  2  79                                          HFU:PU          24 0  0  0  0  33 93                                          0.8:1           48 0  0  0  0  63 98                                                          0  100                                                                              0  0  0  0  0                                           Phytase 5,000 HPU                                                                             1  38 12 47 (+)                                                                              0  15                                          pH 2.5 Acid Phosphatase 5,000 HPU                                                             2  17 7  76 (+)                                                                              6  27                                                          8  0  0  7  19 12 69                                          HFU:PU          24 0  0  0  0  76 99                                          4.4:1           48 0  0  0  0  101                                                                              104                                                         0  100                                                                              0  0  0  0  0                                           Phytase 1,000 HPU                                                                             1  40 16 39 (+)                                                                              0  10                                          pH 2.5 Acid Phosphatase 9,000 HPU                                                             2  27 8  26 (+)                                                                              1  17                                                          8  7  2  21 17 9  45                                          HFU:PU          24 0  0  6  5  46 79                                          36.4:1          48 0  0  0  0  102                                                                              103                                         __________________________________________________________________________     .sup.a Yield, % of theoretical: the starting concentration of 10 mM           Naphytate substrate (IP6) in the assays was considered equal to 100%          theoretical yield (i.e., of IP6); for IP6, the concentration of residual      Naphytate at the end of the assay was divided by the starting                 concentration to obtain the % indicated in Tables 1a and 1b; for IP5, IP4     IP3, Ins, and P, the maximal theoretical concentration of each degradatio     product that could be released from IP6 (i.e., by acid hydrolysis) was        calculated and considered equal to 100% theoretical yield of the              respective degradation product; the concentration of residual degradation     product at the end of the assay was divided by the theoretical                concentration to obtain the % indicated in Tables 1a and 1b; (+) traces. 

                                      TABLE 1b                                    __________________________________________________________________________    pH 2.5          Yield, % of Theoretical.sup.a                                 Enzyme(s)       Time                                                          dosage/mmol substrate                                                                         (h)                                                                              IP6                                                                              IP5                                                                              IP4                                                                              IP3                                                                              Ins                                                                              P.sub.i                                     __________________________________________________________________________                    0  100                                                                              0  0  0  0                                              Finase          1  51 16 24 0  0                                              10,000 PU       2  35 22 29 14 0                                                              8  0  7  1  18 0                                                              24 0  0  0  0  18                                                             48 0  0  O. 0  52 88                                                          0  100                                                                              0  0  0  0                                              Phytase         1  43 33 27 11 0                                              10,000 PU       2  23 18 24 21 0                                                              8  1  2  6  24 0                                                              24 0  0  0  0  0                                                              48 0  0  0  0  0  90                                                          0  100                                                                              0  0  0  0  0                                           pH 2.5 Acid Phosphatase                                                                       1  107                                                                              0  0  0  0  0                                           0 PU            2  106                                                                              0  0  0  0  0                                           10,000 HPU      8  105                                                                              0  0  0  0  0                                                           24 93 0  (+)                                                                              (+)                                                                              0  i                                                           48 96 0  (+)                                                                              (+)                                                                              0  i                                                           0  100                                                                              0  0  0  0  0                                           Phytase 10,000 PU                                                                             1  30 26 45 (+)                                                                              0  18                                          pH 2.5 Acid Phosphatase 1,000 HPU                                                             2  27 23 36 32 0  21                                                          8  I  2  10 29 0  46                                          HFU:PU          24 0  0  (+)                                                                              (+)                                                                              4  82                                          1.4:1           48 0  0  (+)                                                                              (+)                                                                              17 90                                                          0  100                                                                              0  0  0  0  0                                           Phytase 10,000 PU                                                                             1  34 12 65 0  0  21                                          pH 2.5 Acid Phosphatase 5,000 HPU                                                             2  27 22 38 28 0  24                                                          8  1  2  9  27 0  50                                          HFU:PU          24 0  0  0  0  21 88                                          5.4:1           48 0  0  0  0  59 96                                                          0  100                                                                              0  0  0  0  0                                           Phytase 10,000 PU                                                                             1  30 11 54 0  0  24                                          pH 2.5 Acid Phosphatase 9,000 HPU                                                             2  31 22 36 28 24                                                             8  (+)                                                                              2  9  25 9  51                                          HFU:PU          24 0  0  0  0  46 88                                          9.4:1           48 0  0  0  0  102                                                                              102                                         __________________________________________________________________________     .sup.a As in Table 1a, above.                                            

The results presented in Tables 1a and 1b show that the purified phytaseand pH2.5 acid phosphatase enzymes and the Finase commercial enzymemixture catalyzed hydrolysis ofNa-phytate to inositol and inorganicphosphate, but at differing rates and with different pH optima. Underoptimal conditions of pH for each of the purified enzymes (i.e., pH5.0for purified phytase; and pH2.5 for the purified pH2.5 acid phosphatase)approximately equivalent amounts of inorganic phosphate were released in48 hours by all three enzyme preparations (i.e., 90% Pi for phytase; 88%Pi for acid phosphatase; and, 88% or 103% for the Finase mixture).However, the results also show that inositol (Ins, Tables 1a and 1b)production is markedly slower at pH5.0 than at pH2.5. Inositol, as theend product of Na-phytate hydrolysis, could not be detected at pH2.5 norat pH5.0 when purified phytase enzyme is used alone, although IP6, IP5,IP4 and IP3 were completely removed during the hydrolysis time. It canbe concluded that the probable hydrolytic end products of the purifiedphytase enzymes are inositol di-and/or monophosphates. In contrast,purified acid phosphatase catalyzed degradation of phytate (i.e.,IP6=inositolhexaphosphates) at pH2.5, producing inositol as an endproduct. These combined results suggested that in a commercialmixture ofphosphatases, such as Finase, phytase alone is not sufficient to enablecomplete conversion of phytate to inositol and inorganic phosphate.Rather, the results suggested that inositol end products may be derivedfrom the action of enzymes with substrate specificity similar to that ofpurified pH2.5 acid phosphatase. The possibility was thereforeconsidered that the products of phytate hydrolysis by phytase (i.e.,IP5, IP4, IP3, IP2, IP 1) might serve as useful substrates for acidphosphatase that would convert the inositolphosphates into free inositoland inorganic phosphate. The results thus suggested a previouslyunsuspected cooperative enzyme activity for phytate degradation betweenphytase and pH2.5 acid phosphatase. To further explore this possibilitythe substrate specificities of the purified phytase and pH2.5 acidphosphatase were evaluated.

The substrate specificities of purified phytase and pH2.5 acidphosphatase were compared at 37° C. using equivalent concentrations ofphytate substrate (i.e., 20 mM) and temperature (i.e., 37° C.). Phytaseactivity was measured at pH5.0 and acid phosphatase activity wasmeasured at pH2.5 using the molybdate phosphate detection systemdescribed in the Materials and Methods section appearing at the end ofEXAMPLE 1. A comparison of the relative activities of the two differentenzymes on different substrates is presented in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Substrate Specificity of Substantially Purified Phytase                       and pH 2.5 Acid Phosphatase                                                                             pH 2.5 Acid                                                         Phytase   Phosphatase                                                         Relative  Relative                                            Substrate       Activity(%).sup.a                                                                       Activity (%).sup.b                                  ______________________________________                                        Phytic acid Na-salt                                                                           100       36                                                  Phytic acid K-Mg-salt                                                                         106       34                                                  Phytic acid Ca-salt                                                                           56        43                                                  Glucose 6-phosphate                                                                           11        62                                                  ATP             7         72                                                  Fructose 1,6-phosphate                                                                        8         107                                                 Fructose 6-phosphate                                                                          1         18                                                  L-a-glycerophosphate                                                                          1         71                                                  p-nitroyhenyl phosphate                                                                       24        100                                                 ______________________________________                                         .sup.a Percent relative activity = "the measured activity calculated as a     % of that obtained with a standard substrate of phytase (i.e., Naphytate      defined as 100%).                                                             .sup.b Percent relative activity = "the measured activity calculated as a     % of that obtained with a standard substrate of acid phosphatase (i.e.,       pnitrophenyl phosphate; defined as 100%)                                 

The results presented in Table 1a, Table 1b and Table 2 show thatpurified phytase, while effective in mediating the hydrolysis of phyticacid, was less effective in catalyzing hydrolysis of other simplerphosphate-containing substrates. In contrast, purified pH2.5 acidphosphatase, while relatively effective in hydrolyzing simplerphosphates, was relatively ineffective in catalyzing hydrolysis ofphytic acid. The results support the notion of a cooperative enzymaticmixture wherein the products of a purified phytase (e.g., inositoldi-and mono-phosphates) might serve as substrates for a purified pH2.5acid phosphatase so that free inositol and inorganic phosphate are theultimate products of the reaction.

"Balanced" enzyme mixtures:

The possibility was next considered that an effective cooperativeenzymatic mixture might be constructed by optimizing the amounts ofpurified phytase and purified pH2.5 acid phosphatases that were mixedtogether in a preparation so that the activity of the two differentenzymes was "balanced", i.e., to achieve cooperative enzyme activity ata particular pH and temperature to give the optimal rate, efficiency,and completeness of phytate degradation to free inositol and inorganicphosphate. Studies were conducted in which mixtures of purified phytaseand pH2.5 acid phosphatase were formulated at defined ratios of the twoenzyme activities, but while such "balanced" mixtures of purifiedphytase and pH2.5 acid phosphatase offered advantages of increased rateof phytate hydrolysis and more complete conversion to free inositol andinorganic phosphate over commercial preparations, it was not consideredcost-effective to purify, standardize, quality control (QC) and qualityassure (QA) such a "balanced" mixture of enzymes for use in mostcommercial processes (e.g., for animal feeds) within desired ranges ofratios of enzyme activities, e.g., pH2.5 acid phosphatase to phytase.

To solve these problems the possibility was next considered thatmolecular techniques might be used to produce recombinant enzymes thatwere of sufficiently high quality to provide uniform standardizedpreparations, and with the necessary cost-efficiency and QC/QAproperties required for commercial products of "balanced" mixtures.

Amino acid sequencing of phytase and pH2.5 acid phosphatase:

In order to obtain internal amino acid sequences of the purifiedproteins, peptide fragments were prepared using TPCK-trypsin (asdescribed, see Materials and Methods, below). In addition, purifiedpH2.5 acid phosphatase was alkylated (i.e., using 4-vinyl pyridine) andthen digested with lysylendopeptidase C, as described (Materials andMethods). The resultant peptides were purified by reverse-phase highpressure liquid chromatography (RP-HPLC) on a C18 RP column (asdescribed in the Materials and Methods section, below). Amino terminalsequencing of purified peptides was conducted using a gas-pulsed liquidphase sequencer, and the released PTH amino acids were analyzed byRP-HPLC (as described, Materials and Methods). Carboxy-terminalsequencing was conducted using kinetic carboxypeptidase Y digestion,PITC derivatization, and RP-HPLC analysis of the PITC amino acids.

The amino acid sequences of the tryptic peptides of phytase and pH2.5phosphatase, as well as the lysylendopeptidase peptides of pH2.5phosphatase are presented in Tables 3 and 4, below. As the resultspresented in Table 3 and 4 indicate, some peptide preparations were notpure. Tables 3 and 4 also present a comparison of the peptide amino acidsequences with the amino acid sequence deduced from nucleotidesequencing of the genes and the cDNA (Example 2, below), i.e., brackets! in Tables 3-4.

The amino terminal sequence of purified Aspergillus niger var. awamoristrain ALKO243 phytase showed N-terminal sequence (i.e., peptide#1081/N-phy Table 3; LAVPAS(R)NQSTXDT) SEQ. ID. NO. 6 similar to, butnot identical with the reported amino terminal sequence in an A. ficuumphytase (i.e., Ullah, 1988; LAVPASRNQSSGDT). One peptide (i.e.,#420/10phy; LYVEMMQ(N)QA(E)Q(T)PLV) SEQ. ID. NO. 8 was similar to, butnot identical in sequence to, a reported internal peptide in an A.ficuum phytase (i.e., Ullah, 1988, MMQCQAEQEPLVRVLVNDRX) SEQ. ID. NO. 9. Carboxyterminal sequencing of phytase gave the sequence XSA-OH. Onepeptide (i.e., #675; Table 3) contained a KDPR SEQ. ID. NO. 10 sequencehomologous with sequence reportedly present in other phosphatases(namely KDPRA; SEQ. ID. NO. 11 Ullah, 1991).

                                      TABLE 3                                     __________________________________________________________________________    Amino acid sequence of isolated peptides of phytase                           Peptide Nos                                                                            Amino Acid Sequence.sup.a  Amino Acid Sequence Deduced from DNA               sequence!.sup.b                                                      __________________________________________________________________________    #132/12 phy                                                                            Tyr-Tyr-Gly-His(Leu)-Gly-Ala-Gly-Asn-Pro-Leu-Gly-Pro-Thr-Gln                  (SEQ ID NO.12)                                                                 Tyr-Tyr-Gly-His----Gly-Ala-Gly-Asn-Pro-Leu-Gly-Pro-Thr-Gln!(SEQ              ID NO.13)                                                            #133     Thr-Gly-Tyr-Val-Gln(Asn)-Tyr-Val-Gln-Met-(Gln) (SEQ ID NO.14)                  not found in DNA!                                                   #242/1 phy                                                                             Ala-Gln-Pro-Gly-Gln-Ala-Ala-Pro-Lys (SEQ ID NO.15)                             Ala-Gln-Pro-Gly-Gln-Ser-Ser-Pro-Lys!(SEQ ID NO.16)                  #420/10 phy                                                                            Leu-Tyr-Val-Glu-Met-Met-Gln-(Asn)-Gln-Ala-(Glu)-Gln-(Thr)-Pro-Leu             -Val (SEQ ID NO.8)                                                            Leu-Tyr-Val-Glu-Met-Met-Gln-Cys-Gln-Ala-Glu-Gln-Glu-Pro-Leu-Val!(             SEQ ID NO.17)                                                        #410/13 phy                                                                            Phe-Ile-Glu-Gly-Phe-Gln-Ser-Asp-Lys (SEQ ID NO.18)                             Phe-Ile-Glu-Gly-Phe-Gln-Ser-Asp-Lys!(SEQ ID NO.18)                  #416/7 phy                                                                             Tyr-Ala-Phe-Leu-Lys (SEQ ID NO.18)  Tyr-Ala-Phe-Leu-Lys! (SEQ ID              NO.19)                                                               #659/6 phy                                                                             Gly-Leu-Ser-Phe-Ala-Arg (SEQ ID No.20)  Gly-Leu-Ser-Phe-Ala-Arg!              (SEQ ID NO.20)                                                       #670 & #796/2 phy                                                                      Val-Ile-Ala-Ser-Gly-Glu-Lys (SEQ ID NO.21)  Val-Ile-Ala-SAer-Gly-             Glu-Lys!(SEQ ID NO.21)                                               #418/3 phy                                                                             Phe-Tyr-Gln-Arg (SEQ ID NO.22)  Val-Ile-Ala-Ser-Gly-Glu-Lys!(SEQ              ID NO.21)                                                            #785/11phy                                                                             Phe-Tyr-Gln-Arg {=#418 above (SEQ ID NO.22)}, and                             Asp-Ser-Phe-Val-Arg (SEQ ID NO.23)                                   (not pure)                                                                              Asp-Ser-Phe-Val-Arg! (SEQ ID NO.23)                                 #248 (not pure)                                                                        Val/Tyr-Leu/Glu-Val/Ser-Asn/Leu-Asp/Gln (SEQ ID NO.24)  not                   possible to compare to DNA!                                          #784/9 phy                                                                             Tyr-Glu-Ser-Leu-Thr-Arg (SEQ ID NO.25)  Tyr-Glu-Ser-Leu-Thr-Arg!              (SEQ ID NO.25)                                                       #675 (not pure)                                                                        Ser-Ala-Ala-Ser-Leu-Asn-Ser (SEQ ID NO.26) {a fragment of the                 trypsin enzyme}                                                               Leu-Lys-Asp-Pro-Arg (SEQ ID NO.27)  Leu-Lys-Asp-Pro-Arg!(SEQ ID               NO.27)                                                               #783 (not pure)/                                                                       Val-Ile-Ala-Ser-Gly-Glu-Lys (SEQ ID NO.21) {amount = #670 and                 796, above}                                                          4 phy    Tyr-Pro-Thr-Glu-Ser-Lys (SEQ ID NO.28)  Tyr-Pro-Thr-Glu-Ser-Lys!     #244 (not pure)                                                                        Tyr/Asp-Phe/Pro-Asn/Ala-X/X-Gly  not possible to compare to                   DNA!                                                                 #793     Leu-Glu-Asn/Pro-Asp/Phe-Leu-Asp/Ser-Gly/Leu-Phe/Val-Thr-Leu)                  (SEQ ID NO. 30)                                                               Leu-Glu-Asn-Asp-Leu-Ser-Gly-Val-Thr-Leu-Thr! (SEQ ID NO.31)          #792 (double                                                                           Tyr-Tyr-Gly-His-Gly-Ala-Gly-Asn-Pro-Leu-Gly-Pro-Thr-Gln-Gly-Val-G             ly-Tyr-Ala-(SEQ ID NO.32)                                            sequence)/15 phy                                                                       Asn-Glu-Leu-Ile-Ala                                                           {=#132 (half of above} and Val-Tyr-Phe-Ala-Gln-Val-Leu-Ser (SEQ               ID NO.33)                                                                     From this double sequence, the following sequences can be                     deduced                                                                       Val-Thr-Phe-Ala-Gln-Val-Leu-Ser (SEQ ID NO.34)                                 Val-Thr-Phe-Ala-Gln-Val-Leu-Ser! (SEQ ID NO.34)                              and Tyr-Tyr-Gly-His-Gly-Ala-Gly-Asn-Pro-Leu-Gly-Pro-Thr-Gln-Gly-V             al-Gly-Tyr-                                                                   Asn-Glu-Leu-Ile-Ala (SEQ ID NO.32)                                   #800/13 phy                                                                            Phe-Ile-Glu-Gly-Phe-Gln-Ser-Thr (SEQ ID NO.35)                                 Phe-Ile-Glu-Gly-Phe-Gln-Ser-Thr! (SEQ ID NO.35)                     #797/14 phy                                                                            Asp/Asn-Tyr-Leu-Gln-Ser-Leu-Lys) (SEQ ID NO.36)                               !Asp-Tyr-Leu-Gln-Ser-Leu-Lys! (SEQ ID NO.37)                         #795 (Odd behavior                                                                     Asn-Ile-Glu-Pro-Phe-Gln-Val-Asn (SEQ ID NO.38)                       in peptide                                                                              not found in DNA sequence.!                                         sequencing)                                                                   #799/8 phy                                                                             Val-Leu-Val-Asn-Asp-Arg (SEQ ID NO.38) {=#248,                                above}  Val-Leu-Val-Asn-Asp-Arg! (SEQ ID NO.39)                      #1081/N-phy                                                                            Leu-Ala-Val-Pro-Ala-Ser-(Arg)-Asp-Gln-Ser-Thr-X-Asp-Thr (SEQ ID               NO.6)                                                                          Leu-Ala-Val-Pro-Ala-Ser-Arg-Asn-Gln-Ser-Thr-Cys-Asp-Thr! (SEQ                ID NO.40)                                                            C-terminal/C phy                                                              (Arg)-Ser-Ala-OH  Cys-Ser-Ala-End!                                            __________________________________________________________________________     .sup.a peptide sequence,                                                      X = amino acid not detected;                                                  / = either one or the other of the two indicated amino acids may be           present, the assay was not definitive,                                        () = the presence of the amino acids in parentheses is subject to questio     beacuase of a weak signal of the PTHamino acid; phytase peptides designte     (phy) were obtained by tyrptic digestion;                                     .sup.b  ! =  peptide sequence deduced from DNA sequence, Example 2,           below!; and,                                                                  #: peptides number.                                                      

In sequencing of pH2.5 phosphatase, no results were obtained from aminoterminal sequencing of either native or alkylated proteins purified fromAspergillus niger var. awamori strain ALKO243. One peptide (i.e.,#1107T/7Lpho, Table 4, below) yielded a sequence that was identical toan amino terminal sequence reported in a phosphatase purified from A.ficuum (i.e., Ullah & Cummins, 1987; FSYGAAIPQSTQEKQFSQEFRDG)(SEQ. ID.NO. 4. Peptide #941C/10Lpho from purified pH2.5 phosphatase (ALKO243)may be a continuation of the #1107/7Lpho sequence, since it appears tobe included in the A. ficuum N-terminus. The peptide #1112T/3Tpho (SEQ.ID. NO. 42 ) seems to be a continuation of peptide #943C/11Lpho, sinceit appears to have a partially overlapping sequence (i.e., FSSG in#1112T/3Tpho). The pH2.5 phosphatase peptide #816C/1Lpho contains apossible active site consensus sequence (i.e., RHGXRXP)(SEQ. ID. NO. 43

                                      TABLE 4                                     __________________________________________________________________________    Amino Acid Sequence of Isolated Tryptic (T) and Lysylendopeptidase (C)        Peptide.sup.a                                                                 of pH 2.5 Phosphatase                                                         Peptide Nos                                                                              Amino acid sequencing.sup.b  DNA sequencing!.sup.c                 __________________________________________________________________________    #816C/1 Lpho                                                                             Arg-His-Gly-Glu-Arg-Tyr-Pro-Ser-Pro-Ser-Ala-Gly-Lys (SEQ ID                   NO.44)                                                                         Arg-His-Gly-Glu-Arg-Tyr-Pro-Ser-Pro-Ser-Ala-Gly-Lys! (SEQ ID                 NO.44)                                                             #817C and 1107T/7                                                                        Phe-Ser-Tyr-Gly-Ala-Ala-Ile-Pro-Gln-Ser-Thr-Gln-Glu-Lys (SEQ                  ID NO.45)                                                          Lpho        Phe-Ser-Tyr-Gly-Ala-Ala-Ile-Pro-Gln-Ser-Thr-Gln-Glu-Lys! (SEQ                ID NO.45)                                                          #847C/5 Lpho                                                                             Asp-Ile-Glu-Glu-Ala-Leu-Ala-Lys (SEQ ID NO.46)                                 Asp-Ile-Glu-Glu-Ala-Leu-Ala-Lys! (SEQ ID NO.46)                   #826C (not pure)                                                                         Ser/Ala-Ile-Glu/Pro-(Glu) (SEQ ID NO.47)                                       not possible to compare to DNA!                                   #943C/11 Lpho                                                                            Ala-Arg-Tyr-Gly-His-Leu-Trp-Asn-Gly-Glu-Thr-Val-Val-Pro-Phe-Phe               -Ser-                                                                         Ser-Gly (SEQ ID NO.48)                                                         Ala-Arg-Tyr-Gly-His-Leu-Trp-Asn-Gly-Glu-Thr-Val-Val-Pro-Phe-Ph               e-Ser-                                                                        Ser-Gly! (SEQ ID NO.48)                                            #938C (not pure)/2 Lpho                                                                  (Ser/Arg)-Tyr/His-Gly-Gly/Glu-Asn/Arg-Gly/Tyr-Pro-Tyr/Ser-(Pro)               -Glu/Ser-                                                                     (Ala)-(Gly) (SEQ ID NO.49)                                                     only part of sequence compared to DNA sequence!                               Tyr-Gly-Gly-Asn-Gly-Pro-Tyr! (SEQ ID NO.50)                       #941C (not pure)/10                                                                      Gln-Phe-Ser-Gln-Glu-Phe-X-Asp-Gly-Tyr-(Arg) (SEQ ID NO.51)                    {Predominant species}                                              Lpho        Gln-Phe-Ser-Gln-Glu-Phe-Arg-Asp-Gly-Tyr! (SEQ ID NO.52)                      (Thr/His)-Tyr-Gly-Gly-Asn-Gly-X-Tyr/Pro (SEW ID NO.53)                        {-#938C, above} {Minor                                                        species}                                                           #1106T and #1112T/3                                                                      Phe-Ser-Ser-Gly-Tyr-Gly-Arg (SEQ ID NO.54)                         Tpho        Phe-Ser-Ser-Gly-Tyr-Gly-Arg! (SEQ ID NO.54)                       #1108T (not pure)/9                                                                      Val-Ala-Phe-Gly-Asn-Pro-Tyr) (SEQ ID NO.55) and                               (Asp/Glu)-Leu-Asn-Ala-Ile-Leu-                                     Tpho       Phe/Lys (SEQ ID NO.56)                                             from the sequence above the following sequences were deduced:                            Val-Ala-Phe-Gly-Asn-Pro-(Tyr) (SEQ ID NO.55) and                              (Asp/Glu)-Leu-Asn-Ala-Ile-Leu-                                                Phe/Lys (SEQ ID NO.56)                                                         Val-Ala-Phe-Gly-Asn-Pro-Tyr! (SEQ ID NO. 55) and  not found                  in DNA sequence!                                                   #1110T (not pure)/6                                                                      Asp-Ile-Glu-Glu-Ala-Leu-Ala-Lys {=#847C, above (SEQ ID NO.46)}                and Gln-Leu-Pro-Gln-                                               Tpho       Phe-Lys (SEQ ID NO.57)  Gln-Leu-Pro-Gln-Phe-Lys! (SEQ ID                      NO.57)                                                             #1111T/4 Tpho                                                                            Val-Ser-Tyr-Gly-Ile-Ala (SEQ ID NO.58)  Val-Ser-Tyr-Gly-Ile-Ala               ! (SEQ ID NO.58)                                                   __________________________________________________________________________     .sup.a T: = trypsin digestion;                                                C: = lysylendopeptidase C digestion of 4vinylpyridine treated pH 2.5          phosphatase;                                                                  .sup.b peptide sequence,                                                      X = amino acid not detected;                                                  / = either one or the other of the two indicated amino acids may be           present, the assay was not definitive,                                        () = the presence of the amino acids in parenthesis is subject to questio     because of a weak signal of the PTHamino acid; phosphatase peptides (pho)     obtained by lysylcndopeptidase digestion are identified (L), alkylated        phosphatase peptides obtained by trypsin digestion are identified (T);        and,                                                                          .sup.c  ! =  peptide sequence deduced from DNA sequence, Example 2,           below!; and,                                                                  #peptide number.                                                         

From these amino acid sequences one peptide sequence from phytase (i.e.,#420, Leu Tyr Val Glu Met Met Gln (Asn) Gln Ala (Glu) Gln (Thr) Pro LeuVal (SEQ. ID. NO. 16) with the questionable (Asn) corrected to Cys;Ullah, 1988; Table 3) and two from pH2.5 phosphatase (i.e., #816C; ArgHis Gly Glu Arg Tyr Pro Ser Pro Ser Ala Gly Lys (SEQ. ID. NO. 62)and#1110T; Gln Leu Pro Gln Phe Lys (SEQ. ID. NO. 62), Table 4) wereselected as being useful for the preparation of degenerateoligonucleotide probes for molecular cloning, as described below inExample 2.

Materials and Methods:

Phytase and pH2.5 acid phosphatase enzyme assays: Molybdate detectionsystem:

Both assays measure the amount of inorganic phosphate that is releasedby enzyme action as colorimetrically quantifying using reduction of aphosphomolybdate complex. One phytase unit (PU) is the amount of enzymewhich liberates, under the conditions described below, 1 nmol ofinorganic phosphate from Na-phytate in one minute at 37° C. One pH2.5acid phosphatase unit (HFU) is the amount of enzyme which liberates,under the conditions indicated below, 1 nmol of inorganic phosphate fromP-nitrophenyl phosphate in one minute.

Phytase activity was determined by adding 1 ml of substrate (1% sodiumphytate freshly prepared daily Sigma #P3168!, in 0.2M citrate buffer,pH5.0) to 1 ml of diluted enzyme supernatant to initiate the hydrolysisof orthophosphate. After exactly fifteen minutes at 37° C., thehydrolysis reaction was terminated by the addition 2 ml of 15% TCA(trichloroacetic acid, Merck #807) followed by mixing, cooling, andcentrifugation to remove any precipitate that forms. Releasedorthophosphate was measured by the addition of an equal volume offreshly prepared Reagent C (i.e., 3 volumes of 1M sulfuric acid mixedwith 1 volume of 2.5% (w/v) ammonium molybdate (Merck #1182) and 1volume of 10% (w/v) ascorbic acid (Merck #127) to a 1:10 dilution of thehydrolysis reaction mixture (above). The Reagent C mixture was incubatedat 50° C. for twenty minutes. The absorbances were measured at 820 nmagainst a reagent blank and known standards (1:100-1:400 dilutions of9.0 mM KH₂ PO₄ : Merck #4873) that were used to construct a standardcurve. The amount of phosphate liberated by phytase was used tocalculate phytase in the following manner: namely, the A_(820nm) valuefor the phytase was compared with the A_(820nm) values of the phosphatestandard curve and after correcting for any dilution factors the phytaseactivity was obtained by dividing the phosphorus concentration (nmol/ml)by the hydrolysis time (i.e., 15 min).

pH2.5 acid phosphatase activity was determined in a similar fashion:namely, 0.1 ml of diluted enzyme (diluted in 0.2M Glycine-HCl buffer,pH2.5) was added to 1.9 ml of substrate (30 mM p-nitrophenyl phosphateBoehringer Mannheim! dissolved in 0.2M Glycine --HCl buffer) pH2.5.After a fifteen-minute incubation at 37° C., the reaction was terminatedby addition of an equal volume of 15% TCA (as above). The releasedorthophosphate was measured using Reagent C (as described above), andthe activity determined by a comparison to known diluted phosphatestandards (as above) and by using the calculations described above.

Preparation of tryptic peptide fragments for amino acid sequencing:

Purified phytase (70 μg) in 50 mM Tris-HCl pH7.9 was digested with 2%(w/w) trypsin (TPCK-treated, Sigma) for 2 h at 37° C. and then with afurther 2% (w/w) trypsin for 21 h. Purified pH2.5 acid phosphatase in100 mM Tris-HCl pH8.0 was treated with 2% (w/w) trypsin for 20 h at 37°C. and then with a further 2% (w/w) trypsin for 6 h. The peptideswerepurified as described below.

Preparation of lysylendopeptidase C peptide fragments for amino acidsequencing:

Purified pH2.5 acid phosphatase was alkylated using 4-vinylpyridine asfollows: To lyophilized pH2.5 acid phosphatase (75 μg) was added 40 μl0.5M Tris-HCl pH7.5 containing 6M guanidium hydrochloride, 2 mM EDTA and34 mM DTT. After addition of 1 μl 4-vinylpyridine (Sigma), the reactionmixture was kept at room temperature (22° C.) for 1 h. The reaction wasstopped by addition of 10 ml 1.4M DTT. Alkylated phosphatase was thenpurified on HPLC with a C-1 reverse-phase column (TSK TMS 250; 0.46×4cm) using a 20% to 70% ACN/0.06% TFA gradient (80% to 30% 0.1% TFA) in30 min. The fractions absorbing at 218 nm were pooled and evaporated ina Speed-Vac vacuum centrifuge. The dried sample was then resuspended byadding 60 μl 70 mM Tris-HCl pH9.1 and digested with 2% (w/w)lysylendopeptidase C (Wako Chemicals) for 2 h at 37° C. After additionof a further 2% (w/w) lysylendopeptidase C, the incubation at 37° C. wasprolonged to 26 h. The peptides were purified as described below.

Peptide purification and amino terminal sequencing:

The peptides obtained from enzymatic digestion (above) were separated byHPLC on a C-18 reverse-phase column (Vydac 218 TP B5; 0.46 ×25 cm) witha 90 min gradient from 0 to 60% ACN/0.06% TFA (100 to 40% 0.1% TFA).Absorbance at 218 nm was used for detection of peptides. Amino terminalsequencing of the purified peptides, as well as the native proteins, wasdone by degrading them in a gas-pulsed liquid-phase sequencer (Kalkkinen& Tilgmann, 1988). The released PTH-amino acids were analyzed on-line byusing narrow-bore reverse-phase HPLC.

Carboxy terminal sequencing of phytase:

A lot of purified phytase (53 μg) was digested with carboxypeptidase Y(Sigma, 0.6 U) in 50 mM sodium acetate pH5.6 containing 10% urea and0.05% SDS at room temperature (22° C.). Samples of the digestion werewithdrawn at various time points. These were dried in a Speed-Vac vacuumcentrifuge and derivatized with phenylisothiocyanate (PITC) according tothe amino acid analyzing kit Pico-Tag (Waters association). Analysis ofthe derivatized amino acids was performed by reverse-phase HPLC with thePico-Tag C-18 column, and quantified by identically derivatized aminoacid standards.

EXAMPLE 2 Isolation And Characterization Of Phosphatase Genes PhytaseAnd pH2.5 Acid Phosphatase

General materials and methods are described in the section entitled"Materials and Methods, " which follow the example.!

For molecular cloning of the phytase and the pH2.5 acid phosphatasegenes interior peptides from the enzymes (as described above) wereprepared from A. niger var. awamori strain ALKO243. The genomic DNAencoding phytase and pH2.5 acid phosphatase were cloned and cDNA wasalso cloned so that the complete coding sequence could be determined.Finally, expression vectors were constructed (as described in Example 3,below) that secreted each of the enzymes in a functional form, and adual-gene-transformed strain was also selected that synthesized andsecreted the desired cost-effective and "balanced" mixture of enzymes ata ratio of pH2.5 acid phosphatase to phytase confering upon the mixturethe property of cooperative enzyme activity that is desirable in aparticular commercial use, e.g., in animal feeds.

Design of oligonucleotide probes:

Several internal peptide fragments were sequenced from purified phytaseand pH2.5 acid phosphatase, Example 1, above. A degenerateoligonucleotide complementary to all eight possible codon combinationsof a chosen region (brackets, Table 3) of an internal phytase peptidewas synthesized using the Pharmacia Gene Assembler Plus: namely, thepeptide from phytase with the sequence Leu Tyr Val Glu Met Met Gln CysGln Ala Glu Gin (SEQ. ID. NO. 19) (i.e., peptide #420, Table 3 with thequestionable (Asn) corrected with Cys; Ullah, 1988). PHY-1, shown inTable 5, is a 17-base oligonucleotide mixture which contains one perfectmatch out of eight combinations.

A 17 mer degenerate oligonucleotide, PHY-31, was designed from an acidphosphatase peptide: namely, peptide #816C from pH2.5 phosphatase (i.e.,Arg His Gly Glu Arg Tyr Pro Ser Pro Ser Ala Gly Lys(SEQ. ID. NO. 66)).Through the incorporation of a neutral inosine, one perfect match out of64 possible combinations exists in PHY-31. The nucleotide sequence ofoligo PHY-31 and corresponding peptide sequence is shown in Table 5.PHY-34 is a 17 mer mixture with 128-fold degeneracy constructed tocoding sequence for peptide #1110T of pH2.5 acid phosphatase; PHY-35 isa 17 mer mixture with 64-fold degeneracy also constructed to codingsequence for peptide #1110T. Both PHY-34 and PHY-35 are necessary forcomplete representation of Peptide #1110T. Peptide #1110T is derivedfrom a trypsin digestion of purified native pH2.5 acid phosphatase(Table 4, above).

                                      TABLE 5                                     __________________________________________________________________________    Oligonucleotide Probes for Phytase and pH2.5 Phosphatase                      Enzyme  Amino Acid Sequence.sup.a                                                                             Oligonucleotide Probe                         __________________________________________________________________________    Phytase Peptide #420 (SEQ. ID. NO: 59):                                                                       Oligo PHY-1:                                          Leu Tyr Val Glu  Met Met Gln Cys Gln! Ala Glu Gln                                                     3'- CAT CAT GTT ACG GTT CG -5' (SEQ. ID.                                      NO: 60)                                                                       3'- CAT CAT GTC ACA GTC CG -5' (SEQ. ID.                                      NO: 61)                                       pH2.5 Acid P'ase                                                                      Peptide #816C (SEQ. ID. NO: 44):                                                                      Oligo PHY-31:                                         Arg  His Gly Glu Arg Tyr Pro! Ser Pro Ser Ala Gly                                                     3'- GTG CCG CTC GCI ATG GG -5' (SEQ. ID.                                      NO: 62)                                                                       3'- GTA CCA CTT TCI ATA GG -5' (SEQ. ID.                                      NO: 63)                                                                       3'- GTG CCG CTC GCI ATG GG -5' (SEQ. ID.                                      NO: 64)                                                                       3'- GTG CCC CTC GCI ATG GG -5' (SEQ. ID.                                      NO: 65)                                               Peptide #1110T (SEQ. ID. NO: 57)                                                                      Oligo PHY 34:                                          Gln Leu Pro Gln Phe Lys!                                                                             5'- CAA CTG CCG CAA TTT AA -3' (SEQ. ID.                                      NO: 66)                                                                       5'- CAG CTA CCA CAG TTC AA -3' (SEQ. ID.                                      NO: 67)                                                                       5'- CAA CTT CCT CAA TTT AA -3' (SEQ. ID.                                      NO: 68)                                                                       5'- CAA CTC CCC CAA TTT AA -3' (SEQ. ID.                                      NO: 69)                                                                       Oligo PHY 35:                                                                 5'- CAA TTA CCG CAA TTT AA -3' (SEQ. ID.                                      NO: 70)                                                                       5'- CAG TTG CCA CAG TTC AA -3' (SEQ. ID.                                      NO: 71)                                                                       5'- CAA TTA CCT CAA TTT AA -3' (SEQ. ID.                                      NO: 72)                                                                       5'- CAA TTA CCC CAA TTT AA -3' (SEQ. ID.                                      NO: 73)                                       __________________________________________________________________________     .sup.a.)  ! = region of sequence utilized in construction of degenerate       oligonucleotides; peptides as described in Tables 3 and 4, above.        

Probing Genomic ALKO243 DNA With Degenerate Oligonucleotides:

In order to evaluate the specificity of the degenerate oligonucleotides,total genomicDNA from ALKO243 was probed with PHY-1 end labelled withλ-³² P!-ATP to a high specific activity using E. coli Polynucleotide T4kinase BRL!. FIG. 7 shows that with a high stringency wash, a uniqueband hybridized to phytase oligo PHY-1. At this stringency, a singleband was recognized by the degenerate oligo which made it a goodcandidate for library screening.

FIG. 7. A. niger var. awamori strain ALKO243 genomic DNA probed withphytase oligonucleotide PHY-1. Genomic DNA was isolated by a neutrallysis method. Briefly, finely ground frozen dried mycelia was lysed witha 4% SDS-TE buffer. Cell debris was removed and supernatant was removedand extracted twice with an equal volume of Tris-saturatedphenol:chloroform (1:1). Genomic DNA was precipitated with NH₄ OAC andEtOH. Pelleted DNA was purified by ultracentrifugation through CsCl asrecovered as described by Maniatis et al. Hybridization to genomic DNAwith (λ³² P) ATP labelled degenerate oligos (Maniatis et al., 1982) wasdone at 42° C. overnight on filters in oligo hybridization solution(6×SSPE, 0.5% SDS, 3×Denhardts, 100 μg/ml tRNA). Non-specific bindingwas removed by washing the filters twice for 30 minutes at roomtemperature with 2×SSC, 0.1% SDS, and once for 5 minutes at 42° C. infresh solution. Overnight exposure on Kodak X-Omat AR film withintensifying screens revealed positively hybridizing bands

Likewise, genomic DNA from ALKO243 was probed with radiolabelledspecific pH2.5 acid phosphatase oligo PHY-31 (under the conditionsdescribed in FIG. 7, above). The autoradiogram is shown in FIG. 8.Although the specificity of PHY-31 was not as high as with PHY-1, itappeared as if, at most, two bands were recognized by thisoligonucleotide probe.

Isolation and characterization of the phytase gene:

Genomic DNA was partially digested with Sau3A in order to producefragments 10-23 kb in length. Digested DNA was ligated to BamHI cutdephosphorylated Lambda Dash II vector arms Stratagene!. The ligationwas packaged in vitro using Stratagene's Gigapack Gold packagingextracts. Packaged phage was used to infect E. coli strain P2392 toobtain plaques. 5×10⁴ plaque forming units were screened with phytaseoligo PHY-1 using the following conditions: namely, 42° C. overnight onfilters in oligonucleotide hybridization solution (6×SSPE, 0.5% SDS,3×Denhardts, 100 μg/ml tRNA). Non-specific binding was removed bywashing the filters twice for 30 minutes at room temperature with 2 XSSC, 0.1% SDS, and once for 5 minutes at 42° C. in fresh solution.Overnight exposure on Kodak X-Omat AR film with intensifying screensrevealed positively hybridizing plaques. Twelve strongly hybridizingplaques were picked for further characterization. Lambda phage withinserts that hybridized to the PHY-1 probes, and had an identical sizeto genomic DNA fragments on 0.8% agarose gel electrophoresis, werechosen for subcloning and eventual nucleotide sequencing (as describedbelow, Materials and Methods).

Subcloning and sequencing of phytase genomic clones:

Bacteriophage DNA isolated essentially by the method of Yamamoto (1970)from each of the 12 candidates was digested with restrictionendonucleases and probed with the PHY-1 oligonucleotide. A 1.8 kbBamHI/Xbal hybridizing fragment previously identified in genomicSoutherns was isolated from clone number CH7 and subcloned into M13mp18and M13mp19 BRL!. Subclone CH7 that reacted positively witholigonucleotide probes for phytase (i.e., PHY-1) was sequenced.

The nucleotide sequence of this subclone revealed regions correspondingto the reported nucleotide sequence of other known phytase peptidesequences, thus confirming the probable identity of the clone as aphytase genomic DNA. Continued sequencing of an overlapping 2.6 kb SphIfragment revealed an open reading frame of 1409 bp that included 15internal peptide sequences and N-terminal peptide sequence. Analysis ofupstream sequence with the IntelliGenetics' PC/GENE program "SIGNAL"(based on the method of Standen, 1984), revealed a strong eukaryoteKozak consensus sequence followed by a methionine initiation codon.However, this ATG was out of frame with respect to the remainder of thesequence. A potential 102 bp intron delineated by consensus fungaldonor, lariat and acceptor sequences (Rambosek and Leach, 1987) wasidentified between the potential initiation codon and the N-terminalpeptide. Splicing of this putative intron restored the reading framebetween the proposed ATG and the N-terminal peptide amino acid sequence.By incorporating this single putative intron in the 5' end of thephytase gene, the entire polypeptide was encoded by 470 amino acids.Sequence and translation of the phytase gene is shown in FIG. 9.

FIG. 9. Nucleotide sequence from the 2.6 kb SphI fragment including thephytase gene with deduced translation. The proposed intron donor(GTRNGT)(SEQ. Id. NO. 74), lariat (RCTRAC)(SEQ. ID. NO. 75) and acceptor(YAG) consensus sequences are overlined. The nucleotide sequencecorresponding to peptide #420 (Table 3) is underlined. The nucleotidesequence was determined by the M13-dideoxy method (Sangeretal., 1977) ofoverlapping subclones with the use of the United States BiochemicalSequenase II kit.

The relative molecular mass of the translated phytase polypeptide wascalculated at approximately 51,400 daltons. A codon usage analysis ofphytase revealed a frequency of G+C at silent third position of sensecodons (i.e., excluding the Trp and Met codons) of 68.3%. The entirestructural gene and upstream sequence was subcloned as a 2.6 kb SphIfragment into pUC-18 and designated pFF-1 (FIG. 10).

FIG. 10. Circular transformation vectors of phytase. (A) pFF-1: a 2.6 kbSphI fragment containing the phytase gene and its native promoter wassubcloned from a positive lambda clone into pUC-18. An XbaI site wasintroduced at the -26 position of the phytase coding region in pFF-1 bysite directed mutagenesis to give pSELFX. (B) pFF-2: phytase undercontrol of the A. niger β-tubulin promoter. A 2.0 kb XbaI fragment frompSELFX was ligated to a unique XbaI site in the fungal expression vectorpTL-113 to give pFF-2. (C) pFF-3: phytase under control of the A. nigerGAPDH promoter. The 2.0 kb XbaI fragment from pSELFX was ligated to aunique XbaI site in pPRE-81 to give pFF-3. (D) pFF-4: phytase undercontrol of the A. niger GA promoter. The 2.0 kb XbaI fragment frompSELFX was ligated to a unique XbaI site in pGA to give pFF-4.

Isolation and characterization of the pH2.5 acid phosphatase gene:

The same lambda library was re-plated and screened with pH2.5 acidphosphatase oligonucleotide PHY-31 using conditions established withgenomic hybridizations above. Twelve hybridizing plaques were picked forfurther characterization. Bacteriophage DNA isolated from each of thecandidates was digested with restriction endonucleases and probed witheither the PHY-31 oligo, or a second oligo mixture, PHY-34/35 that wasderived from an independent pH2.5 acid phosphatase peptide (Table 5).One of the clones, AP99, contained a 2.1 kb SphI fragment previouslyidentified in genomic Southern analysis, that hybridized strongly toboth oligonucleotides. Strong hybridization to two oligonucleotidesderived from independent peptide sequence strongly suggested that theclone contained pH2.5 acid phosphatase sequence. This fragment wassubcloned into M13mp18 and M13mp19 for sequencing. The nucleotidesequence of this subclone revealed an ORF of 785 bp with a potential 5'initiation ATG (methionine), a fungal signal sequence, and a translatedsequence compatible with the N-terminal peptide and other internalpeptide sequences determined in Example 1, above (Table 4). Downstreamof the ORF, termnination codons were identified in all three readingframes until nucleotide 1151, after which an additional pH2.5 acidphosphatase peptide sequence was identified. These results necessitatedthe inclusion of an intron(s) in the 3' portion of the gene.

Identification of cDNA clones for pH2.5 acid phosphatase:

PolyA mRNA was purified (as described, below; Materials and Methods) andused for polymerase chain reaction (PCR) cloning of a portion of pH2.5acid phosphatase cDNA. Briefly, first strand synthesis was performedwith the oligonucleotide primer AP273 which was annealed to subspeciesmRNA encoding pH2.5 acid phosphatase: namely, AP2735-CTACCCCTCTGCATCTAG-3' (SEQ. ID. NO. 76). Oligonucleotide PCR primersUPPHOS and DOWNPHOS were synthesized with flanking EcoRI restrictionsites: namely,

UPPHOS 5'-GAATTCCGAGTCCGAGGTCATGGGCGCG-3(SEQ. ID. NO. 77); and,

DOWNPHOS 5'-GAATTCCCGGGACCTACCCCTCTGCAT-3(SEQ. ID. NO. 78).

UPPHOS and DOWNPHOS were inversely oriented and separated by 978 basesin genomic clones. PCR amplification of the cDNA-mRNA complex witholigonucleotide primers UPPHOS and DOWNPHOS yielded a specific productof approximately 850 bps. The amplified product was isolated fromagarose gels and digested with EcoRI. This fragment was then subclonedinto pUC-18 digested with EcoRI for double stranded sequencing.

PCR-amplified cDNA from the 3' portion of the gene was sequenced andrevealed the presence of three short introns, each exhibiting consensusfungal donor, lariat and acceptor sequences. The exon sequence isderived by splicing the nucleotides 136-916, 971-1088, 1141-1245 and1305-1740 (as shown in FIG. 11). The resultant translated sequence codesfor a protein of 479 aa. The entire sequence with translation is shownin FIG. 11.

The deduced pH2.5 acid phosphatase polypeptide has a calculatedmolecular weight of approximately 52,700 daltons. A codon usage analysisof pH2.5 acid phosphatase revealed a very high frequency of G+C atsilent third position of sense codons of 79.3%.

The 2.1 kb SphI fragment contained only 135 bp of upstream pH2.5 acidphosphatase sequence. Because this length of promoter sequence may nothave been enough for efficient expression, a larger fragment wassubcloned from the lambda clone. pAP-3 contains a 5.1 kb SalIl/PstIfragment from lambda clone AP99 and the nucleotide sequence is shown inFIG. 11.

FIG. 11. Nucleotide sequence from the 2.1 kb SphI fragment containingthe pH2.5 acid phosphatase gene with deduced amino acid translation. Theintron donor, lariat and acceptor sequences as determined by cDNAsequencing are overlined. The nucleotide sequence corresponding topeptides #816 and #1110 (Table 4) is underlined. The genomic nucleotidesequence was determined by the M13-dideoxy method (Sanger et al., 1977)with the use of the United States Biochemical Sequenase II kit.

Materials and Methods:

Enzymes:

Restriction enzymes were purchased from Bethesda Research Laboratoriesand New England Biolabs (Beverly, Mass., USA). T4 DNA ligase, T4 DNApolymerase, T4 kinase, and the Klenow fragment from E. coli DNApolymerase I were purchased from BRL (Gaithersburg, Md., USA). Calfintestine phosphatase (CIP) was purchased from Boehringer Mannheim(Indianapolis, Ind., USA). Spheroplasting enzyme, Novozyme, waspurchased from Novo Biolabs (Bagsvaerd, Denmark). All enzymes were usedin accordance with the manufacturer's recommendations. Enzyme assaysubstrates phytic acid and para-nitrophenylphosphate (PNPP) werepurchased from Sigma (St. Louis, Mo., USA) and BMB, respectively.

Bacterial and fungal strains, plasmids and phage:

A. niger var. awamori strain ALKO243 (ATCC#38854) was used for isolationof the genes for phytase and pH2.5 acid phosphatase.

E. coli strains LE392 and P2392 and the phage Lambda Dash II, used inthe construction of the A. niger gene bank, were obtained fromStratagene (La Jolla, Calif., USA). E. coli strain JM109 was used as ahost in transformations with constructions derived from the plasmidspUC18 and pUC19. The phage M13mp18 and M13mp19 used in thedideoxynucleotide sequencing were obtained from Bethesda ResearchLaboratories (Gaithersburg, Md., USA).

Phleomycin resistant vector pLO-3, which contains the phleomycinresistance gene (a phleomycin binding protein gene fromStreptoalloteichus hindustanus) coupled to a yeast cytochrome C1terminator, was derived from the plasmid pUT713 (CAYLA, Toulouse Cedex,France). It is expressed in fungus by the β-tubulin promoter of A.niger.

General growth media:

E. coli JM109 was grown in L-broth. Transformants were selected onL-plates supplemented with 1.5% agar and containing 125 μg/mlampicillin. Complete medium (CM) for growth of fUngus in liquid iscomposed of: 50 ml of 20×Clutterbuck's salts (120 g NaNO₃, 10.4 g KCl,10.4 g MgSO₄ 7H₂ O, 30.4 g KH₂ PO₄), 2.0 ml Vogel's Trace Elements (0.3Mcitric acid, 0.2M ZnSO₄, 25 mM Fe(NH₄)₂ (SO₄)₂ ·6H₂ O, 10 mM CuSO4, 3 mMMnSO₄ ·H₂ O, 8 mM boric acid, 2 mM Na₂ MoO₄ ·2H₂ O), 5.0 g tryptone, 5.0g yeast extract, 10 g glucose, in one liter of distilled water. A. nigerstrains ALKO243 and ALKO2268 were grown on PD agar slants (2.4% PotatoDextrose Broth Difco #0549!; 1.5% Agar Difco #0140!) for seven to twelvedays at 28° C.

Isolation of genomic DNA from A. niger var. awamori strain ALKO243:

One slant of ALKO243 grown on PDA for 5 days at 35° C. was soaked with 5ml of NP-40 H₂ O (0.005% (v/v) Nonidet P40 detergent). Spores werescraped from slants andmacerated in glass tubes with 3 mm glass beads.1×10⁸ spores were used to inoculate 500 ml CM in a 2-liter flask.Cultures were grown at 35° C. with shaking at 200 RPM for 48 hours.Mycelia were collected on mira-cloth, frozen in liquid nitrogen andlyophilized overnight. All of the frozen dried mycelia (approximately2.0 grams) were ground with sea sand in a mortar chilled with liquidnitrogen. Ground mycelia were transferred to a 250 ml centrifuge bottleand resuspended in 30 ml 4% SDS-TE buffer (10 mM Tris Base, 1 mM EDTA,4% SDS) and allowed to lyse at room temperature for 1 hour. Cell debriswere removed by centrifugation at 4000×g for 5 minutes and thesupernatant was removed to a 30 ml centrifuge tube. The samples wereextracted twice with an equal volume of Tris-saturated phenol:chloroform(1:1) each time removing the aqueous phase to a clean tube. DNA wasprecipitated by adding 10% (v/v) 5M NH₄ OAc and 2.5 volumes of EtOH, andincubating overnight at -80° C. The preparation was thawed at roomtemperature until "syrupy" and was centrifuged at 12000×g for 30 minutesat 4° C. The supernatant was removed and the pellet dissolved in 19 mlTE (10 mM Tris Base, 1 mM EDTA) with gentle pipeting, to which was added19.0 g CsCl. Two 11.5 ml ultracentrifuge tubes were filled with DNA inTE+CsCl, 0.9 ml of 10 mg/ml ethidium bromide was added and the mixturewas centrifuged at 45,000 RPM (20° C.) for 22 hours in a Sorvall T865.1rotor. Banded genomic DNA was visible under UV light and collectedthrough an 18 m gauge hypodermic needle. Ethidium was removed byextraction with NaCl saturated isopropanol. CsCl was removed by dialysisagainst TE. DNA was precipitated with ethanol in 0.3M NaOAc.

Construction of genomic gene bank:

Genomic DNA from A. niger var. awamori strain ALKO243 was cut with Sau3Ain order to produce fragments 10-23 kb in length. Cut DNA was ligated topurchased BamHI cut dephosphorylated Lambda Dash II vector arms. Theligation was packaged in vitro using Stratagene Gigapack Gold packagingextracts. Packaged phage was used to infect E. coli strain P2392 toobtain plaques.

Screening the gene bank with oligonucleotides:

Plaques were lifted onto Schleicher & Schuell NC (BA85) nitrocellulosemembranes as recommended by the manufacturer. Using the amino acidsequences from phytase and pH2.5 acid phosphatase (Example 1, above),degenerate oligonucleotides were prepared for each protein. Theoligonucleotide mixture for each protein was complementary to allpossible codon combinations of the chosen region of the peptide.Oligonucleotides were synthesized using the Pharmacia Gene AssemblerPlus and the sequences are shown in Table 5, above.

Isolation of Lambda DNA:

Single isolated hybridizing plaques were picked into 500 ml SM (perliter: 5.8 g NaCl, 2.0 g MgSO₄, 50 ml 1M Tris-HCl, pH7.5; 5 ml 2% (w/v)gelatin solution). 20 ml chloroform was added and phage particles wereeluted overnight at 4° C. 220 ml of cell lysate was mixed with 200 mlLE392 cells grown in the presence of Mg and maltose; 8 ml NZCYM media(10.0 g NZ-amine, 5.0 g NaCl, 5.0 g yeast extract, 2.0 g MgSO₄ ·7H₂ O,and 1.0 g Casamino acids per liter; pH adjusted to pH7.5) was added;and, the culture was grown with shaking at 37° C. until cells lysed(about 6 hours). 100 ml chloroform was then added and incubation wascontinued for 15 minutes to ensure complete lysis. The sample was thencentrifuged 5 min at 8000 g and the supernatant was removed to freshtube. RNAse A and DNAse I were added to 1 mg/ml each; the samples wereincubated for 30 minutes at 37° C; and, an equal volume of 20% (wv) PEG8000, 2M NaCl in SM was added to precipitate the phage. Samples wereincubated for at least 1 hour on ice. The resultant phage were pelletedby centrifuging 10,000×g for 20 minutes at 4° C.; the supernatant wasdecanted; and, the phage pellet was resuspended in 0.5 ml SM. DNA waspurified further by addition of SDS, EDTA and Proteinase K, followed byextraction with phenol:chloroform and isopropanol precipitation. Thesizes of the lambda DNA inserts were then examined on 0.8% agarose gels,the DNA was blotted to nitrocellulose and hybridized with theradiolabelled oligonucleotide PHY-1 or PHY-31 probes. DNA fragments thathybridize to the probes and had a size identical to genomic DNAfragments were chosen for subcloning.

Isolation of total RNA from A. niger:

Three 200 ml cultures of A. niger var. awamori ALKO243 were grown in RNAbroth media (2% corn starch Sigma!, 1% proteose peptone Difco!, 30 g/lglucose, 5 g/l NH₄ NO₃, 0.5 g/l MgSO₄ ·7H₂ O, 0.5 g/l KCl, 0.183 glFeSO₄ ·7H₂ O) for 48 hours at 30° C., 220 RPM. The mycelia were filteredthrough Whatman filter paper #1 and rinsed with 10 ml of DEPC-treated H₂O (0.1% diethyl pyrocarbonate in sterile distilled water), thenlyophilized overnight at -80° C. 1.5 grams (3 g total) of dried myceliawas crushed under liquid nitrogen into a fine powder, then transferredto a centrifuge tube containing 10 ml of Breaking Buffer (50 mM Tris-HC1pH7.4, 150 mM NaCl, 5 mM EDTA pH8, 5% SDS) and 10 ml ofphenol/chloroform/isoamyl alcohol (P/CIA: 50:48:2). The mixture wasallowed to thaw for thirty minutes at room temperature and was thencentrifuged for 10 minutes at 10,000 RPM at 4° C. The aqueous layer wasremoved using a wide bore pipette into a clean centrifuge tube andre-extracted with P/CIA until the interface was clear. An equal volumeof 6M LiCl was added to the final aqueous layer to precipitate the RNAand chilled overnight at -80° C. The mixture was then centrifuged fortwenty minutes at 10,000×g and 4° C., and the pellet was resuspended in2.4 ml of GTC solution (4M Guanidine thiocyanate, 25 mM Sodium citratepH7, 5% N-lauryl sarcosine, 100 mM b-mercaptoethanol). An equal volumeof isopropanol was added, the precipitate was chilled on dry ice for 40minutes, and then centrifuged at 4° C. for 30 minutes. The pellet wasrinsed in 80% EtOH, re-spun, vacuum dried, and finally resuspended in 1ml of DEPC-H₂ O. The concentration of RNA was determinedspectrophotometrically at 260 nm.

Isolation of mRNA:

Polyadenylated messenger RNA (polyA mRNA) was affinity purified fromtotal RNA using oligo(dT)-cellulose columns according to themanufacturers instructions (Pharmacia Fine Chemicals, Piscatawy, N.J.).Briefly, 1.25 mg of total RNA was applied to two separate columns thatwere previously subjected to centrifugation and equilibration inHigh-salt buffer (Pharmacia). After three washes in Low-salt buffer(Pharmacia), the mRNA was eluted from the column at 65° C. in prewarmedElution Buffer (10 mM Tris-HCL, pH7.4, containing 1 mM EDTA); andprecipitated by adding 0.1 volume of 10 mg/ml glycogen and 2.5 volumesof ethanol. The mixture was chilled at -80° C. for 2 hours, thencentrifuged at 4° C. for 30 minutes. The recovered MRNA was dissolved inElution Buffer to a final concentration of 1.3 mg/ml.

PCR isolation of pH2.5 acid phosphatase cDNA:

First strand synthesis was performed with the BMB cDNA kit according tomanufacturers' recommendations with 1.0 mg mRNA and oligonucleotideprimer AP273, synthesized complementary to 3' pH2.5 phosphatasenucleotide sequence: namely, AP273 5'-CTACCCCTCTGCATCTAG-3'(SEQ. ID. NO.76). Oligonucleotide PCR primers UPPHOS and DOWNPHOS were synthesizedwith flanking EcoRI restriction sites: namely,

UPPHOS 5'-GAATTCCGAGTCCGAGGTCATGGGCGCG-3'(SEQ. ID. NO. 77); and,

DOWNPHOS 5'-GAATTCCCGGGACCTACCCCTCTGCAT-3'(SEQ. ID. NO. 78),

as described above.

Subcloning and sequencing of phytase and pH2.5 acid phosphatase clones:

Hybridizing restriction fragments of lambda genomic clones were gelpurified using the "Glassmilk Purification Kit" available commerciallyfrom GeneClean (Bio 101, La Jolla, Calif.). The restriction fragmentswere subcloned into M13mp-18 and M13mp-19 cut with the appropriateenzymes. The nucleotide sequence of clones reacting positively witholigonucleotide probes for phytase and pH2.5 acid phosphatase wasdetermined by the M13-dideoxynucleotide sequencing method (Sanger etal., Proc. Nat. Acad Sci. USA 74:5463-5467, 1977) using the UnitedStates Biochemical Sequenase II kit. cDNA was sequenced in regions ofsuspected introns by alkaline denaturation double stranded sequencing.The phytase gene was completely contained within a 2.6 kb SphI fragmentthat was subcloned into pUC-18 to give pFF-1. The complete pH2.5 acidphosphatase gene was isolated as a 2.1 bp SphI fragment that wassubcloned into pUC-18 to give pAP-1.

pSELFX, is a plasmid containing a phytase nucleotide sequence in whichan XbaI site was introduced into the gene sequence by modifying thenucleotides at position -26 and -24 relative to the ATG start codon. Thenucleotide changes were introduced by site-specific mutagenesis of the2.6 kb SphI fragment containing the phytase gene, using the PromegaAltered Sites Mutagenesis Kit. The oligonucleotide used for directingmutagenesis is shown below: namely, ##STR1##

pAP-1Xba is a plasmid containing a pH2.5 acid phosphatase nucleotidesequence in which an XbaI site was introduced into the gene sequence bymodifying the nucleotides at positions -24 and -27 relative to the ATGstart codon. The nucleotide changes were introduced by PCR-mutagenesis.The primers for PCR-mutagenesis are shown below: namely, ##STR2##

pAP-1 DNA was used as a template for PCR amplification. The resultingamplified fragment was cleaved with XbaI and NruI and the fragment wasthen ligated to XbaI/NruI cut pAP-1 to give plasmid pAP-1Xba. Theplasmid was sequenced to insure that no mutations were introduced intothe region which had been PCR amplified. pAP-2 was generated by ligatingthe BamHI/HindIII fragment treated with Klenow reagent from pAP-1XBAinto ,SmaI cut pUC-18. Transformants containing the correctly orientedpAP-2 were identified by analysis of the size of restrictionendonuclease fragments.

The entire structural gene for pH2.5 acid phosphatase was removed frompAP-2 as a 2.0 kb XbaI fragment for construction of pH2.5 acidphosphatase transformation vectors that over-expressed the subjectenzyme (Example 4, below). The complete pH2.5 acid phosphatase gene wasalso isolated as a 5.0 kb PstI/SalI fragment that was subcloned intopUC-18 to give pAP-3. pAP-3 was used in the construction of pH2.5 acidphosphatase transformation vectors for over-expressing the subjectenzyme (Example 4, below).

Northern blotting procedure:

Ten and twenty mg amounts of total RNA (isolated as above) were appliedto wells of a 1.5% agarose-formaldehyde gel and separated byelectrophoresis. RNA molecular weight markers (0.24 kb-9.5 kb "RNALadder," Bethesda Research Labs) were also applied to the gel for sizecomparisons. The gel was blotted onto nitrocellulose (Schleicher andSchuell; Keene, NH), baked for one hour at 80° C. in a vacuum oven,prehybridized in 50% formamide, 5X SSC, 0.1% SDS, 5×Denhardt's and 100mg/ml calf thymus DNA at 37° C. for 24 hours, then hybridized in thesame solution with radiolabelled probe at 42° C. for 24 hours. Thefilter was washed twice in 2×SSC/0.1% SDS at room temperature forfifteen minutes, then once in 0.1×SSC/0.1% SDS and autoradiographedusing Kodak S-Omat AR film with intensifying screens.

EXAMPLE 3 Homology With Other Phosphatases

A comparison was made between the amino acid sequence of the pH2.5 acidphosphatase and phytase and other published acid phosphatase sequences.Homology (alignment score of 17.705) was determined using the PC/GENEmatrix program PCOMPARE (based on the method of Neddleman and Wunsch,1970). Significant homology was found between the pH2.5 acid phosphataseand other acid phosphatase enzymes "PHO-3" (Bajwa, et al., 1984) and"PHO-5" (Arima, et al., 1983), both isolated previously by others fromSaccharomyces cerevisiae. The area of highest homology, R H G X R X P(SEQ. ID. NO. 82)(aa positions 81-87), was used to search the EMBLCDPROT17 protein database release 1991. The RHGXRXP (SEQ. ID. NO. 82)sequence was found in several other acid phosphatases (as shown in Table6,

                                      TABLE 6                                     __________________________________________________________________________    Homology of phytase and pH2.5 Acid Phosphatase                                with Other Acid Phosphatase.sup.a                                             Organism                                                                           Gene Name.sup.b                                                                     Amino Acid Sequence                 SEQ ID NO.                     __________________________________________________________________________    A. niger                                                                           Phytase                                                                             A Q V L S R H G A R Y P T E S K G K (SEQ ID NO. 83)                A. niger                                                                           pH2.5 AP                                                                            V I M V K R H G E R Y P S P S A G    K                                                                        (SEQ ID NO. 84)                    E. coli                                                                            APPA  V V I V S R H G V R A P T K A T Q    L                                                                        (SEQ ID NO. 85)                    Yeast                                                                              PHO-1 V H T L Q R H G S R N P T G G N A    A                                                                        (SEQ ID NO. 86)                    Yeast                                                                              PHO-5 L Q M V G R H G E R Y P T V S L A    K                                                                        (SEQ ID NO. 87)                    Yeast                                                                              PHO-3 L Q M L A R H G E R Y P T Y S K G    A                                                                        (SEQ ID NO. 88)                    Rat  LAP   V T L L Y R H G D R S P V K A Y P    K                                                                        (SEQ ID NO. 89)                    Rat  PAP   V T L V F R H G D R G P I E T F P    N                                                                        (SEQ ID NO. 90)                    Human                                                                              ACP2  V T L L Y R H G D R S P V K T Y P    K                                                                        (SEQ ID NO. 91)                    Human                                                                              ACPP  V T L V F R H G D R S P I D T F P    T                                                                        (SEQ ID NO. 92)                    __________________________________________________________________________     .sup.a.) Homology with other acid phosphatases found using the target         sequence R H G X R X P with IntelliGenetics PC/GENE program QGSEARCH on       the EMBL CDPROT17 protein database using default search parameters; BOLD      residues are identical;                                                       .sup.b.) APPA = A pH2.5 acid phosphatase from E. coli ( Touati and            Danchin, 1987!; PHO1 = An acid phosphatase from Scizosaccharamyces pombe      ( Elliot et al., 1986!); PHO5 = repressible acid phosphatase from             Saccharomyces cerevisiae ( Arima et al., 1983!); PHO3 = nonrepressible        acid phosphatase from S. cerevisiae ( Bajwa et al., 1984!); LAP = rat         lysosomal acid phosphatase ( Himeno et al., 1989!); PAP = a rat prostatic     acid phosphatase ( Roiko et al., 1990!); ACP2 = a human lysosomal acid        phosphatase ( Pohlmann et al., 1988!); ACPP = a human prostatic acid          phosphatase ( Tailor et al., 1990!).                                     

The RHGXRXP region is conserved between organisms as diverse as E. coliand humans. This conservation suggests functional significance and thusmay reflect an active site region of these phosphatases.

EXAMPLE 4 Over-Expression Of Recombinant Phytase And ph2.5 AcidPhosphatase In Aspergillus Niger

General materials and methods are described in the section entitled"Materials and Methods," which follows this example.!

Construction of expression vectors phytase:

A number of vectors were designed for the reintroduction of phytase andpH2.5 acid phosphatase using both native and alternative fungalpromoters. One set of vectors carrying the phytase gene, pFF1-pFF4 (FIG.10, above), contained no fungal selectable marker and were introduced byco-transformation with the plasmid pLO3 (see Materials and Methods,below).

Recombinant strains resulting from vectors pFFI-2 contained foreign DNAsequences derived from the E. coli selectable marker. Therefore, asecond set of vectors termed pFF6-pFF11 was designed to remove thesesequences. The latter vectors contained a fungal selectable marker andrestriction sites allowing linear fragments to be isolated free of E.coli sequences (FIG. 12).

FIG. 12. Linear transformation vectors of phytase. (A) pFF-6A: phytaseunder control of its native promoter. The 2.6 kb SphI fragment frompFF-1 was cloned into the SphI site of pLO3 to give pFF-6, was isolatedas a linear 4.9 kb HindIII fragment. (B) pFF-8: phytase under control ofthe GAPDH promoter. The phleomycin resistance cassette from pLO3 wasisolated as a HindIII (filled)/PstI fragment and subcloned into BglII(filled)/Pst cut pPRE-81 to give pPRE-82. The pSELFX XbaI fragmentcontaining phytase was added to pPRE-82 partially cut with XbaI to givepFF-8, that was isolated as a 6.7 kb PstI fragment. (C)pFF-9: phytaseunder control of the GA promoter. The phleomycin resistance cassettefrom pLO3 was isolated as a KpnI (filled)/HindIII fragment and ligatedto pFF-4 partially cut with KpnI, blunted, then cut with HindIII. pFF-9was isolated as a 7.3 HindIII/KpnI linear fragment. (D)pFF-11: phytaseunder control of the GA promoter and signal sequence. Oligos #260 and#261 (see Materials and Methods, below) were annealed and ligated toXbaI/XhoI digested pFF-1, the GA promoter added as a KpnI/XbaI fragment,and the phleomycin resistance marker (Phleo^(r)) cassette was added as aHindIII fragment to give pFF-11. Linear DNA was isolated as a 7.35 KpnIfragment.

Construction of expression vectors: pH2.5 acid phosphatase with andwithout phytase:

Transformation vectors for pH2.5 acid phosphatase consisted of linearvectors, termed pPH01-pPH04A, that were constructed for introduction ofthe pH2.5 acid phosphatase gene (FIG. 13). Two vectors, pFIN-1A andpFIN-1B, were also engineered to contain both the phytase and pH2.5 acidphosphatase genes expressed from the glucoamylase promoter in a singleplasmid (FIG. 14).

FIG. 13. Vectors from which linear fragments can be isolated for thereintroduction of pH2.5 acid phosphatase. (A) pPHO-1: pH2.5 acidphosphatase under control of its native promoter. The phleo^(r) cassettewas isolated as a HindIII fragment, blunt-ended, and ligated to pAP-3(the pH2.5 acid phosphatase gene in a 5.0 kb PstI/SalI fragment inpUC-18 (5)), cut with EcoRI and also blunt-ended. Linear DNA wasisolated as a 6.3 kb PstI fragment. (B) pPHO-2: pH2.5 acid phosphataseunder control of the b-tubulin promoter. The pH2.5 acid phosphatase genecontained in a 2.0 kb XbaI fragment from pAP-1Xba (Materials andMethods, below) was subcloned into pTL-113, which was then partially cutwith SphI, filled, and ligated to a fragment containing thephleomycin-resistance marker cassette, yielding pPHO-2. (C) pPHO-3:pH2.5 acid phosphatase under control of the GAPDH promoter. pPHO-2 waspartially digested with XbaI to give a 4.3 kb XbaI fragment containingpH2.5 acid phosphatase and the phleo^(r) cassette, which was the ligatedinto the XbaI site of pPRE-81. (D) pPHO-4A: pH2.5 acid phosphatase undercontrol of the GA promoter. The 2.0 kb Xba fragment from pAP-1Xba wassubcloned into pGA, and a 5.5 kb Kpnl fragment containing the GApromoter and pH2.5 acid phosphatase was isolated and filled in and thenligated into pLO3.

FIG. 14. A dual-enzyme-transformation vector was constructed forover-expression of both phytase and pH2.5 acid phosphatase by a singleplasmid. (A) pFIN-1A and (B) pFIN-1B: combination plasmids with bothphytase and pH2.5 acid phosphatase under control of separate copies ofthe GA promoter. An 8.1 kb HindIII fragment was isolated from pPHO-4Aand ligated into HindIII-digested pFF-4.

Transformation vector constructs: general considerations:

As is evident from the information in the discussion above of FIGS.12-15, several different fungal promoter constructions were evaluated:namely, the β-tubulin promoter, theglyceraldehyde 3-phosphatedehydrogenase promoter (GAPDH), and the glucoamylase promoter (GA). Thelatter two fungal promoters were isolated from A. niger ATCC1015(Tailor, P. et al. 1990). These respective promoters were tested todetermine their ability to drive expression of phytase and pH2.5 acidphosphatase.

To facilitate fusion of alternative promoters into the constructs, anXbaI restriction site was introduced by site-directed mutagenesis at the-26 position of the phytase gene. Similarly, an XbaI site was engineeredat position -28 of the pH2.5 acid phosphatase gene by PCR mutagenesis(see Materials and Methods, below). The engineered Xbal sites weresubsequently used for fusions to alternative fungal promoters previouslyengineered to contain such XbaI sites in the 5' regulatory regions ofthe gene.

The results obtained in cells transformed with phytase vector constructsare presented first, below, followed by the results obtained with pH2.5acid phosphatase.

Transformation of A. niger strains ALKO243 and ALKO2268:

A. niger var. awamori strain ALKO243 and A. niger strain ALKO2268, amutant strain exhibiting higher production of phytase derived fromALKO243 by UV-mutagenesis, were used as hosts for engineered phytase andpH2.5 acid phosphatase. A. niger strains ALKO243 and ALKO2268 weretransformed by a modification of the procedure of Tilburn et al. (1983)using phleomycin resistance as a drug-selectable genetic marker. Becausethe cloned phytase and pH2.5 acid phosphatase genes were maintained onplasmids without a selectable marker, spheroplasts were co-transformedwith a phleomycin resistant vector pLO-3 that contains the phleomycinbinding protein gene from Streptoalloteichus hindustanus coupled to ayeast cytochrome C1 terminator. pLO-3 was derived from the plasmidpUT713 CAYLA, Toulouse Cedex, France! and is expressed in fungus by theβ-tubulin promoter of A. niger . Transformants were selected with anequal volume overlay containing 50 μg/ml phleomycin for strain ALKO243and 195-200 μg/ml for strain ALKO2268. (Strains ALKO243 and ALKO2268were both sensitive to spheroplast lysis, and required different osmoticbuffering conditions; see Materials and Methods, below.) Thirty toforty-five sporulating colonies were obtained per μg of DNA on selectionplates. All phleomycin resistant colonies had integrated the phleomycinresistance marker (Southern analysis, data not shown).

Screening of transformants for over-expression of phytase:

Transformants producing higher phytase activity than parental A. nigerstrain ALKO243 were identified in a phytase plate screening assay thatcolorimetrically measures the accumulation of inorganic phosphate by thereduction of a phosphomolybdate complex (Materials and Methods, below).Transformants with high activity in the plate assay were subsequentlytested for phytase production by assaying the enzyme activity releasedinto the broth of a fermentation culture (as described, below).

FIG. 15 shows a plate assay for detecting increased phytase activity.Conidia from A. niger phytase transformants GAD-10 through GAD-120 wasspotted onto plate assay media, incubated for two days at 30° C., andoverlaid with Reagent C for color detection (Materials and Methods,below). Positives GAD 130 (a4), GAD 112 (e3) and GAD 118 when grown inshaker flask cultures, exhibited titers of 108,000 PU/ml, 35,600 PU/ml,and 36,700 PU/ml, respectively (i.e., increases of about up to about1260-fold higher than ALKO243). Phytase activity for untransformedALKO2268 (b5) was 4,000 PU/ml.

Analysis of phytase over-producing transformants:

The amount of phytase produced by transformants was analyzed by growingthe cells in shaker flask cultures and titering the amount of enzyme inthe broth, as follows: briefly, cultures were established in shakerflasks under optimal conditions (Materials and Methods, below) andincubated for 5 days, at which time the cells were removed bycentrifugation and the supernatant broth from the culture was titratedto determine the amount of phytase activity. (Both the plate assay(above) and the broth titer assay measured the amount of inorganicphosphate released by enzymatic hydrolysis of sodium phytate substrateby colorimetrically quantifying the reduction of a phosphomolybdatecomplex.)

Using the native phytase promoter (from plasmid; pFF-1 see, Example 2,Materials and Methods, above), of the 58 transformants generated, fourexhibited increased production of phytase that was 6.5- to 7.0-foldgreater than that produced by non-transformed over-producer strainALKO2268 ("Untransformed", Table 7, below) or by ALKO2268 control cellsthat were transformed with a control plasmid pLO-3 which contained nophytase sequences.

                  TABLE 7                                                         ______________________________________                                        Over-expression of phytase in transformants                                   of production strain ALKO2268.sup.a                                           Strain    Plasmid Promoter   Activity.sup.b                                                                       Increase.sup.c                            ______________________________________                                        ALK02268  --      Native      4,000 --                                        Untransformed                                                                 GAD17     pFF-1   Native     26,000 6.5                                       GAD33     pFF-1   Native     17,500 4.4                                       GAD40     pFF-1   Nafive     10,500 2.6                                       GAD48     pFF-1   Native     28,000 7.0                                       ______________________________________                                         .sup.a Recombinant ALK02268 overproducers of either phytase in shake flas     cultures, i.e., fermentation in a soy flour cultivation media, after 5        days of shaking at 28° C. Cellular and media components were           removed by centrifugation. Enzyme activity was determined by adding           dilutions of the culture broth supernatant to 1% sodium phytate  Sigma!,      in 0.2 M citrate buffer, pH 5.0. After fifteen minutes at 37° C.,      each reaction was terminated by adding 15% TCA (trichloroacetic acid,          Merck!). Released orthophosphate was measured by adding an equal volume      of Reagent C (3:1:1 ratio of 1 M sulfuric acid  Mallinckrodt!, 2.5%           ammonium molybdate  Signma!, 10% ascorbic acid  Sigma!) to a 1:10 dilutio     of the hydrolysis reaction and incubating at 50° C. for twenty         minutes. Absorbance of the reaction mixture was measured against a reagen     blank and known phosphate standards (1:100-1:400 dilutions of 9.0 mM          KH.sub.2 PO.sub.4) at 820 nm;                                                 .sup.b Activity is expressed as phytase units per ml (i.e., PU/ml);           .sup.c Increase is expressed as the fold increase in activity (i.e., in       PU/ml) over the activity (i.e., in PU/ml) with ALK02268 (i.e., activity o     transformant activity of ALK02268).                                      

Further studies involved use of heterologous promoters driving phytaseexpression. 1467 phytase transformants were examined using the plateassay (157 were derived from strain ALKO243, and 1310 were from strainALKO2268). Of these, 302 were identified in the plate assay as beingpotential over-producers of phytase. Of the phytase-over-producingtransformants, 25 (i.e., 1.7% of the 1467 evaluated) had enzyme activityin excess of 700-fold greater PU/ml than that produced by ALKO243. (Theresults obtained with 18 of the 25 transformants are shown in Table 8.)The transformant exhibiting the highest phytase production (i.e.,GAI-6;181,000 PU; about 2,100-fold greater PU/ml than ALKO243) was the resultof transformation with the pFF-9 construct containing a glucoamylase(GA) fungal promoter. The latter transformant showed significantlyincreased levels of phytase production compared to ALKO2268 (anover-producer) or to ALKO243 (a wild-type strain) (see Table 8, below).

                  TABLE 8                                                         ______________________________________                                        A. niger phytase transformants which produced                                 phytase enzyme over 700-fold greater than ALK0243                             in shake flask cultures*                                                                                         Ac-   In-                                  Transformant                                                                           Strain    Plasmid.sup.a                                                                          Promoter                                                                             tivity.sup.b                                                                        crease.sup.c                         ______________________________________                                        GAI-6    A.2268    PFF-4    GA     181,000                                                                             2129                                 GAL-142  A.2268    pFF-3    GAPDH  174,000                                                                             2047                                 GAN-1    A.2268    pFF-3    GAPDH  170,000                                                                             2000                                 GAG-12   ALK0243   pFF-3    GAPDH  166,000                                                                             1953                                 GAO-248  A.2268    pFF-9    GA     164,000                                                                             1929                                 GM42     A.2268    PFF-4    GA     145,000                                                                             1706                                 GAK446   A.2268    pFF-4    GA     132,000                                                                             1553                                 GM-2     A.2268    pFF-4    GA     131,000                                                                             1541                                 GAK4-52  A.2268    pFF-4    GA     131,000                                                                             1541                                 GAM-111  A.2268    pFF-6A   NATIVE 129,000                                                                             1518                                 GAK447   A.2268    pFF-4    GA     121,000                                                                             1424                                 GAM-225  A.2268    pFF-9    GA     112,000                                                                             1318                                 GAD-103  A.2268    pFF-3    GAPDH  108,000                                                                             1271                                 GAD-23   A.2268    pFF-3    GAPDH  108,000                                                                             1271                                 GAM-199  A.2268    pFF-9    GA     107,600                                                                             1266                                 GAE-32   A.2268    pFF-6A   NATIVE  72,600                                                                              854                                 GAL-65   A.2268    pFF-9    GA      72,200                                                                              849                                 GAE-3    A.2268    pFF-6A   NATIVE  67,500                                                                              794                                 Control  ALK02268  None     Native  3,800                                                                               45                                  Control  ALK0243   None     Native    85   1                                  ______________________________________                                         .sup.a Plasmids pFF9, pFF8 and pFF6A were used in linear form with all E.     coli sequences excised.                                                       Transformant designation;                                                     GA = glucoamylase promoter;                                                   GAPDH = glyceraldehyde 3phosphate dehydrogenase promoter;                     NATIVE = native phytase promoter;                                             .sup.b = Activity from shake flask fermentation culture; Enzyme activitie     (i.e., in PU/ml) are averages of two independent fermentations;               .sup.c Increase is expressed as the fold increase in activity in shake        flask culture over the activity with ALK0243 in shake flask culture (i.e.     activity in PU/ml of transformant per activity in PU/ml of ALK0243).     

Molecular characterization: phytase transformants:

Southern and Northern analyses were conducted with DNA and MRNA(respectively), isolated from phytase transformants GAE 32, GAK 4-47,GAL 65, GAM 225 and GAO 248 (Table 8) that over-produced phytase inshake flask fermentation cultures. Genornic DNA was isolated from therespective transformant strains, digested with restriction enzymes, andprobed with a radiolabelled cloned phytase DNA restriction fragment(i.e., isolated from pFF1, above), to determine the gene copy number andthe method of integration in the transformants. An increase in gene copynumber was determined by comparing the density of the blot at 1.3 kb(i.e., the transformation vector construct DNA) against the density ofthe endogenous chromosomal copy of the phytase gene (i.e., at 2.4 kb)and against control ALKO2268 genomic DNA. Quantification was achieved byscanning densitometry (FIG. 16A). Three to five additional phytasevector gene copies, arranged in multiple different patterns ofintegration (data not shown), were detected in transformants GAE 32, GAK4-47, GAL-65, GAM-225 and GAO-248.

Expression of phytase mRNA levels by Northern blotting demonstrated thatmessage levels of transformants were consistently increased by seven- tothirteen-fold over non-transformed controls (FIG. 16B). However, actuallevels may be higher because it proved difficult to isolate mRNA fromcells grown in production media, and it was therefore necessary to makethese comparisons under sub-optimal conditions for cell growth andphytase expression.

FIG. 16A. Southern blot analysis of phytase genomic copy number inover-producing phytase transformants: genomic DNA was digested withBamHI, blotted, electrophoresed, and the filter was probed with BamHIfragments of the 5' region of pFF-1. Gene copy number was determined bycomparing blot density of gene copies (at 1.3 kb) in transformants withthe blot density of the endogenous phytase gene (at 2.4 kb) by scanningdensitometry. Lanes 1, 4, 7: Untransformed control ALKO2268. Lanes 2, 5,8, 9, 10: over-producing phytase transformants GAE-32, GAK-47, GAL-65,GAM-225, GAO-248, respectively.

FIG. 16B. Northern blot analysis messenger RNA levels in phytaseover-producing transfornants: 20 μg of each total RNA sample waselectrophoresed in a formaldehyde gel and transferred to nitrocellulose.Each transformant was probed with the restriction fragments of thephytase gene (i.e., BamHI fragments from pFF-1.). Increased phytasemessage level was measured by scanning densitometry of the mRNA. RNAfrom ALKO2268 (Lane 1) was used as baseline control. Lanes 2-6: GAE-32,GAK-47, GAL-65, GAM-225, GAO-248, respectively.

The results presented in Table 9, below, summarize the quantitativeresults obtained by scanning the density of the Southern and Northernblots in FIG. 16A and FIG. 16B.

                  TABLE 9                                                         ______________________________________                                        Comparison of phytase gene copy number mRNA levels                            and enzyme production by phytase over-producers                                                  Copy                                                       Strain  plasmid.sup.a                                                                            No.    mRNA.sup.b                                                                           Activity.sup.c                                                                       Increase.sup.d                        ______________________________________                                        ALK0243 None       1.0    --        85  1                                     ALK02268                                                                              None       1.0    1.0     3,800   45                                  GAO-248 pFF9 (GA, lin)                                                                           6.0    9.2    164,000                                                                              1,929                                 GAK47   pFF4 (GA, cir)                                                                           3.0    13.2   121,400                                                                              1,428                                 GAM-225 pFF9 (GA, lin)                                                                           3.0    7.6    112,000                                                                              1,318                                 GAE-32  pFF6 (nat, cir)                                                                          6.0    11.3    72,600                                                                                854                                 GAL-65  pFF9 (GA. lin)                                                                           3.0    11.8    72,200                                                                                849                                 ______________________________________                                         .sup.a nat = native promoter;                                                 cir = circular vector;                                                        lin = linear DNA fragment;                                                    GA = glucoamylase promoter;                                                   .sup.b mRNA = messenger RNA amplification levels (as foldincrease over        levels in ALK0243);                                                           .sup.c Activity = phytase production in fermentation culture represented      in phytase units per ml (i.e., PU/ml), conditions described in Table 7,       above;                                                                        .sup.d = Increase is expressed as the fold increase in activity in shake      flask culture over the activity with ALK0243 in shake flask culture (i.e.     activity in PU/ml of transformant per activity in PU/ml of ALK0243).     

Analysis of over-producing pH2.5 acid phosphatase transformants:

It was not possible to develop a plate screening assay specific forpH2.5 acid phosphatase; therefore, transformants were analyzed byevaluating enzyme production in the culture broth after 5 days offermentation in shake flasks (as described above, Table 7, and in theMaterials and Methods, below). The conditions for measuring productionof pH2.5 acid phosphatase in the fermentation shake-flask assay aredescribed below (Table 10) using paranitrophenylphosphate as thesubstrate (also described below in the Materials and Methods). As inTable 7, above, the levels of enzyme in the culture broth supernatantwere titered and the levels of enzyme were compared with the levelsproduced over the same 5-day period of incubation by a control cultureof the ALKO243 strain of A. niger.

Of the 55 transformants generated with the plasmid pAP-3 containing thenative promoter driving expression of pH2.5 acid phosphatase (Example 2,Materials and Methods, above), four transformants showed increases inproduction of pH2.5 acid phosphatase that was 25- to 57-fold greater(Table 10) than the levels produced by parental strain ALKO243. Thehighest producing transformant (GAQ56) exhibited nearly a 58-foldincrease over untransformed ALKO2268 (Table 10), and up to a 126-foldincrease is illustrated in Table 11, below.

                  TABLE 10                                                        ______________________________________                                        Over-expression of pH 2.5 acid phosphatase in                                 transformants of production strain ALK02268.sup.a                             Strain    Plasmid Promoter   Activity.sup.b                                                                       Increase.sup.c                            ______________________________________                                        Untransformed:                                                                A.2268    None    Native      2,300 --                                        Transformed:                                                                  GAQ56     pAP-3   Native     133,000                                                                              57.5                                      GAO13     pAP-3   Native     102,500                                                                              44.5                                      GAQ66     pAP-3   Native      77,800                                                                              33.8                                      GA062     pAP-3   Native      57,600                                                                              25.0                                      ______________________________________                                         .sup.a Conditions, as described in Table 7, above;                            .sup.b Activity in acid phosphatase units per ml (i.e., HFU/ml);              .sup.c Increase is expressed as the fold increase in activity in shake        flask culture over the activity with ALK02268 in shake flask culture          (i.e., activity in HFU/ml of transformant per activity in HFU/ml of           ALK02268).                                                               

Further studies evaluated heterologous promoters driving pH2.5 acidphosphatase expression. 1181 transformants over-produced pH2.5 acidphosphatase (410 resulted from strain ALKO243 and 771 were from strainALKO2268). 32 transformants (2.7%) were identified that produced pH2.5acid phosphatase in excess of 30 HFU/ml. The highest producingtransformant (i.e., GAO-69) averaged 126 HFU/ml. This expressionresulted from transformation with a plasmid construct having a fungalglucoamylase (GA) promoter (Table 11).

                                      TABLE 11                                    __________________________________________________________________________    A. niger pH 2.5 acid phosphatase transformants                                over-producing enzyme at levels over 40-fold higher than ALK0243              in shake flask fermentation cultures.sup.a                                    Transformant                                                                          Strain                                                                              Plasmid                                                                              Promoter                                                                           Activity.sup.b                                                                     Increase.sup.c                                 __________________________________________________________________________    GAO-69  A.2268                                                                              pPREPHO-4                                                                            GA   693,000                                                                            126                                            GAW-131 A.2268                                                                              pPHO-4A                                                                              GA   583,000                                                                            106                                            GBL-128 ALK0243                                                                             pPHO-3 GAPDH                                                                               566,500*                                                                           103*                                          GBL-97  ALK0243                                                                             pPHO-3 GAPDH                                                                               533,500*                                                                           97*                                           GAO-61  A.2268                                                                              pPREPHO-4                                                                            GA   451,000                                                                            82                                             GAW-89  A.2268                                                                              pPHO-3 GAPDH                                                                              412,500                                                                            75                                             GAW-130 A.2268                                                                              pPHO-4A                                                                              GA   385,000                                                                            70                                             GAW-121 A.2268                                                                              pPHO-4A                                                                              GA   379,500                                                                            69                                             GBL-87  ALK0243                                                                             pPHO-3 GAPDH                                                                               346,500*                                                                           63*                                           GBL-119 ALK0243                                                                             pPHO-3 GAPDH                                                                               335,500*                                                                           61*                                           GAO-84  A.2268                                                                              pPREPHO-4                                                                            GA   330,000                                                                            60                                             GAW-54  A.2268                                                                              pPHO-3 GAPDH                                                                              280,500                                                                            51                                             GBL-129 ALK0243                                                                             pPHO-3 GAPDH                                                                               264,000*                                                                           45*                                           GAW-141 A.2268                                                                              pPHO-4A                                                                              GA   258,500                                                                            47                                             GBL-103 ALK0243                                                                             pPHO-3 GAPDH                                                                               242,000*                                                                           44*                                           GAW-112 A.2268                                                                              pPHO-4A                                                                              GA   236,500                                                                            43                                             GBL-92  ALK0243                                                                             pPHO-3 GAPDH                                                                               236,500*                                                                           43*                                           GAW-114 A.2268                                                                              pPHO-4A                                                                              GA   231,000                                                                            42                                             GAT-143 ALK0243                                                                             pPHO-4A                                                                              GA   231,000                                                                            42                                             Control ALK02268                                                                            None   native                                                                              2,300                                                                               0.5                                          Control ALK0243                                                                             None   native                                                                              5,500                                                                              1                                             __________________________________________________________________________     .sup.a Transformed ALK02268 and ALK0243 overproducers of pH 2.5 acid          phosphatase in shake flask fermentation cultures (conducted as described      in Table 7, above). Enzyme activity was determined by adding dilutions of     the culture broth supernatant to 30 mM pnitrophenyl phosphate (Boehringer     Mannheim) in 0.2 M glycineHCL, pH 2.5 buffer. The reaction was terminated     and orthophosphate assayed as described in the footnote to Table 7. Enzym     activities are averages of activities from two independent fermentations      except (*), which were from single fermentations. Plasmids pPHO3 and          pPHO4A were used in linear forin with E. coli sequences excised.              Transformant designation:                                                     GA = glucoamylase promoter;                                                   GAPDH = glyceraldehyde 3phosphate dehydrogenase promoter;                     .sup.b Activity in acid phosphatase units per ml (i.e., HFU/ml);              .sup.c Increase is expressed as the fold increase in activity in shake        flask culture over the activity with ALK0243 in shake flask culture (i.e.     activity in HFU/ml of transformant per activity in HFU/ml of ALK0243).   

Molecular characterization of pH2.5 acid phosphatase transformants:

Copy number and message levels were determined using Southern andNorthern blot analysis and scanning densitometry (as described above)for five of the highest pH2.5 acid phosphatase over-producers identifiedin the shake flask fermentation cultures (Table 11, above). DNA frompH2.5 acid phosphatase transformants GAT-143, GAW-54, GAW-89, GAW-121and GAW-130 was digested with restriction enzymes and hybridized to aradiolabelled pH2.5 acid phosphatase-specific probe. Overall, the pH2.5acid phosphatase transformants had higher gene copy numbers (Table 12,below) than observed above with the phytase transformants (Table 9,above). In contrast, message levels for pH2.5 acid phosphatasetransformants (Table 12) were not as high as observed in phytasetransformants (Table 9). However, as with phytase transformants above,the pH2.5 acid phosphatase message levels were not measured underoptimal fermentation conditions because of the difficulty encountered inisolating RNA from cells under production culture conditions. Therefore,expression under production conditions may be higher than that measuredhere.

                  TABLE 12                                                        ______________________________________                                        Comparison of pH 2.5 Acid Phosphatase Gene Copy Number with mRNA              levels and Enzyme Production by Recombinant Transformed Strains.sup.a                                    pH 2.5                                                                 Copy   Acid Phosphatase                                   Strain Plasmid      No.    mRNA  Activity.sup.b                                                                       Increase.sup.c                        A.2268 None          0     1.0    2,300  0.4                                  ______________________________________                                        GAT-143                                                                              pPHO4A (GA, lin)                                                                           10.0   5.6   231,000                                                                              42.0                                  GAW-54 pPHO3 (GAP, lin)                                                                            7.0   5.7   280,500                                                                              51.0                                  GAW-89 pPHO3 (GAP, lin)                                                                           12.0   3.0   412,500                                                                              75.0                                  GAW-121                                                                              pPHO4A (GA, lin)                                                                           11.0   3.0   379,500                                                                              69.0                                  GAW-130                                                                              pPHO4A (GA, iin)                                                                            7.0   7.2   385,000                                                                              70.0                                  ______________________________________                                         .sup.a GA = glucoamylase promoter;                                            GAP = glyceraldehyde 3phosphate dehydrogenase promoter;                       lin = linear DNA fragment;                                                    mRNA = messenger RNA amplification levels (as foldincrease over levels in     ALKO243);                                                                     .sup.b = pH 2.5 acid phosphatase activity represented in acid phosphatase     units per ml (HFU/ml); comparisons made with cells grown in shake flask       cultures;                                                                     Activity = enzyme activity measured as described in Table 10, above;          .sup.c Increase is expressed as the fold increase in activity in shake        flask culture over the activity with ALK0243 in shake flask culture (i.e.     activity in HFU/ml of transformant per activity in HFU/ml of ALKO243).   

Materials and Methods:

Plasmid constructions were designed so that vector backbone sequencescould be easily removed prior to transformation of the respective genesinto fungi. Linear DNA was isolated from agarose and purified byGeneClean to remove any possible contaminating specificities. ConstructsA-D and Constructs E-H expressing the phytase and pH2.5 phosphatasegenes, respectively, under different regulatory control were prepared asdescribed below.

Phytase and pH2.5 acid phosphatase expression plasmids:

Construct A: pFF-6 phytase under control of its native promoter.

The 2.6kb SphI fragment containing the phytase gene was cloned into theSphI site of pLO-3. Two orientations were generated, termed pFF-6A andpFF-6B. Linear DNA was isolated from either vector as a 4.9 kb HindIIIfragment.

Construct B: pFF-8 Phytase under control of the A. niger GAPDH promoter.

Fungal expression vector pPRE8-1 was cut with BglII and blunted with DNApolymerase I Klenow fragment; the plasmid was then cut with PstI tocompletion. The phleomycin resistance (Phleo^(r)) marker expressioncassette was removed from pLO-3 (as a HindIII (filled)/PstI fragment),and ligated to the cut pPRE8-1 to give pPRE8-2. Transformants wereidentified by restriction analysis. The 2.0 kb XbaI fragment containingthe phytase gene (Example 2, Materials and Methods) was ligated into aunique XbaI site downstream from the GAPDH promoter in plasmid pPRE8-2.Correctly oriented transformants containing pFF-8 were identified byrestriction analysis. Linear DNA was isolated from pFF-8 as a 6.7 kbPstI fragment.

Construct C: pFF-9 Phytase under control of the A. niger GA promoter.

The 2.0 kb XbaI fragment containing the phytase gene (Example 2,Materials and Methods, above) was ligated to the unique XbaI site offungal expression vector pGA to give pFF-4. Transformants containingcorrectly oriented plasmid were identified by restriction analysis.pFF-4 was cut partially with KpnI to produce singularly cut linearplasmid. The ends were blunted with T4 DNA Polymerase. The plasmid wasthen cut to completion with HindIII. pLO-3 was first cut with KpnI andblunted with T4 DNA Polymerase, and then cut with HindIII. The 2.1 kbfragment containing the phleo^(r) expression cassette was purified fromagarose with GeneClean and ligated to cut pFF-4. Transformantscontaining pFF-9 were identified by restriction analysis. Linear DNA wasisolated from pFF-9 as an 7.3 kb HindIII/KpnI fragment.

Construct D: pFF-11 Phytase under control of the A. niger GA promoterwith secretion via the GA signal sequence.

Oligonucleotides 260 and 261 were synthesized coding for an XbaI siteupstream from the translation initiation region and signal sequence forglucoamylase, which was located, in turn, upstream from nucleotidesequence encoding the N-terminus of phytase (SEQ. ID. NO. 1). (Theconstruct also contained a native XhoI site immediately downstream fromthis region.) The two synthetic oligonucleotides used in this constructhad the following nucleotide sequence: namely, ##STR3##

Oligonucleotides 260 and 261 were annealed and then ligated to XbaI/XhoIcut pFF-1 to give pFF-1GA. The glucoamylase promoter from pGA wasligated into pFF-1GA (as a KpnI/XbaI fragment) to give pPREFF-11.Finally, a phleo^(r) cassette was added as a HindIII fragment into aunique HindIII site of the construction. Transformants containingcorrectly oriented plasmid were identified by restriction analysis.Linear DNA was isolated from pFF-11 as a 7.35 KpnI fragment. ConstructE: pPHO-1 pH2.5 Acid Phosphatase (AP) under control of its nativepromoter.

pAP-3 was cut with EcoRI and blunt-ended with Klenow. The phleo^(r)cassette was removed from pLO-3 as a HindIII fragment, blunt-ended withKlenow, and ligated into the cut pAP-3 plasmid. Transformants containingcorrectly oriented pPHO-1 were identified by restriction analysis.Linear DNA was isolated from pPHO-1 as a 6.3 kb PstI fragment.

Construct F: pPHO-2, pH2.5 Acid Phosphatase gene under control of the A.niger b-tubulin promoter.

Fungal expression vector pTL113 was cut with XbaI and dephosphorylatedwith calf intestinal phosphatase. A 2.0 kb XbaI fragment containing thepH2.5 phosphatase gene (Example 2, Materials and Methods, above) wasligated into cut pTl113 to give pPREPHO-2. Transformants containingcorrectly oriented plasmids were identified by restriction analysis.pPREPHO-2 was partially cut with SphI to give singularly cut linearplasmid. The ends of cut pPREPHO-2 were blunted with T4 DNA Polymerase.A 2.7 kb blunt-ended HindIII fragment containing the phleo^(r)expression cassette from pLO-3 was ligated into the cut pPREPHO-2vector. Transformants containing correctly oriented pPHO-2 plasmid wereidentified by restriction analysis. Linear DNA was isolated as a 6.0 kbPstI fragment.

Construct G: pPHO-3, pH2.5 acid phosphatase gene under control of the A.niger GAPDH promoter.

A 4.3 kb XbaI fragment containing the pH2.5 phosphatase gene (Example 2,above) and the phleo^(r) expression cassette was removed from pPho-2 bypartial digestion. The fragment was purified from agarose with GeneCleanand ligated into a unique XbaI site in fungal expression vector pPRE8-1.Transformants containing correctly oriented pPHO-3 plasmid wereidentified by restriction analysis. Linear DNA was isolated as a 7.0 kbPstI fragment.

Construct H: pPHO-4, pH2.5 Acid Phosphatase gene under control of the A.niger GA promoter.

The 2.0 kb XbaI fragment containing the pH2.5 acid phosphatase gene(Example 2, above) was ligated into XbaI cut fungal expression vectorpGA to give pPPEPHO-4. Transformants containing correctly orientedplasmid were identified by restriction analysis. A 5.5 kb Kpnl fragmentcontaining the GA promoter and the pH2.5 Acid phosphatase gene wasremoved from pPREPHO-4 by partial digestion. The resulting fragment waspurified from agarose with GeneClean and blunt-ended with T4 DNAPolymerase. The blunt-ended fragment was ligated into pLO-3 cut withPstI, and then the DNA was blunt-ended to give plasmids in two differentorientations, termed pPHO-4A and pPHO-4B. Transformants containing eachorientation were identified by restriction analysis. Linear DNA wasisolated as an 8.1 kb HindIII fragment.

DNA transformation of A. niger strains ALKO243 and ALKO2268:

Spheroplasts were obtained by first adding 0.5 ml to 1 ml of a fungalspore suspension from conidiating cultures grown on PD slants to 50 mlof CM media in 250-ml flasks. For ALKO243, cultures were grown overnightat 35° C., 200 RPM, before filtration. ALKO2268 cultures were grown 48hours at 30° C., 200 RPM. The resulting mycelia were collected onto adouble layer of cheesecloth, added to 50 ml of KCM buffer (0.7M KCI, 10mM MOPS, pH5.8) with 5 mg/ml Novozym 234 (Novo BioLabs) and incubated at30° C., 85 RPM overnight for spheroplast generation.

The spheroplasts were harvested by filtration through a funnel packedwith mira cloth and covered with cheesecloth into four 15 ml conicalcentrifuge tubes, then spun for ten minutes, 1500 RPM in a bench topcentrifuge. The pellets were gently resuspended in a total of 15 mlSorbitol Buffer (1M Sorbitol, 50 mM CaCl₂) and re-centrifuged. Thepellet was again washed in Sorbitol Buffer (SB) then resuspended in SBto a density of 5×10⁷ /ml.

5 μg of linear or plasmid DNA in 20 μl TE was added to 200 μl ofspheroplasts. For co-transformations 1 μg of selectable phleomycinresistance marker gene DNA, pLO3, was also added. 50 μl of PCM (40% PEG8000, 10 mM MOPS, pH5.8, 50 mM CaCl₂ CaCl₂ added just before use!) wasgently pipetted into the DNA-spheroplast mixture and incubated on icefor 30 minutes.

1 ml of PCM was added to the transformation mix, the mix was pipettedinto 50 ml of Regeneration Agar (MA: CM plus 1.3M mannitol, 3% agar) anddivided into five petri dishes. Spheroplasts were allowed to regenerate3 to 5 hours at 35° C. before overlaying with an equal amount ofOL+phleomycin (OL: 1% peptone, 1% agar; phleomycin CAYLA!: 50 μg/ml forALKO243, 195 μg/ml for ALKO2268). Putative transformants weretransferred to PD+phleomycin slants and grown at 28° C.

Phytase plate assay:

Transformants producing higher phytase yields were identified by a plateassay that colorimetrically measures the accumulation of inorganicphosphate by the reduction of a phosphomolybdate complex. Conidia fromputative transformants was spotted onto assay plates and incubated twodays at 30° C. 20 ml of Reagent C (3:1:1 ratio of 1M sulfuric acidMallinckrodt!, 2.5% ammonium molybdate Sigma!, 10% ascorbic acid Sigma!was applied to the top of the media, and the plates were incubated at50° C. for fifteen minutes before scoring for color intensity. (Plateassay agar: 2% corn starch Sigma #S-4126!, 1% protease peptone Difco#0122-1!, 30 g glucose/1, 5 g NH₄ NO₃ /1, 0.5 g MgSO₄ ·7H₂ O/1, 0.5 gKCl, 0.183 g FeSO₄ ·7H₂ O1, 3% agar, 3% sodium phytate Sigma #P-3168!.)

Phytase and pH2.5 acid phosphatase enzyme assays:

Enzyme assays were conducted as described in Example 1, above.

Production of phytase and pH2.5 acid phosphatase by transformedrecombinant cells:

Transformed recombinant filamentous fungi expressing phytase and pH2.5acid phosphatase were cultivated under identical conditions in asoy-based production medium (50 g/1 soy flour, 30 g/1 glucose, 5 g/1,NH₄ NO₃, 0.5 g/1 MgSO₄ ·7H₂ O, 0.5 g/1KCl, 0.183 g/1FeSO₄ ·7H₂ O, pH5.0)! for five days on a rotary shaker at 200 RPM and 28° C. Transformantsutilizing the glucoamylase promoter were additionally grown in soyglucoamylase media (soy media plus 4% corn starch and 6% glucose. Enzymesamples were collected by centrifuging an aliquot of the fermentationculture at 13,000 RPM in a microcentrifuge for ten minutes, thentransferring the enzyme-containing supernatant to a fresh tube forenzyme assay (below).

EXAMPLE 5 Balanced Over-expression Of Both The Phytase Gene And ThepH2.5 Acid Phosphatase Gene

Analysis of dual-enzyme-transformants with both phytase and pH2.5 APgenes:

Because both pH2.5 acid phosphatase and phytase are required for optimaldegradation of phytic acid, it was considered highly advantageous toconstruct strains that might over-produce both enzymes. Since phyticacid was dephosphorylated most efficiently when ratios of phytase unitsto acid phosphatase units are tailor-made for the specific use (Example1, above), it was considered even more desirable if transformant strainscould be selected that would over-produce both enzymes in this desiredrange of ratio.

A. niger ALKO243 transformants were selected using the combinations oftransformation vector plasmids shown in Table 13 and co-transfectionwith a phleomycin selectable marker (as described above, Example 4,Materials and Methods). Following co-transformation with the indicatedcombinations of phytase and pH2.5 acid phosphatase containing plasmids(Table 13), the transformants were grown in shaker flask cultures (asdescribed in Table 7 and Table 10, Example 4, above) to evaluate enzymeproduction.

All dual-enzyme transformants were first assayed to determine the amountof pH2.5 acid phosphatase; if levels were significantly elevated, thenphytase activity was assayed.(Since phytase has two pH optimums at pH2.5and pH5.0 above, Example 1!, its activity can be detected under pH2.5acid phosphatase parameters.) (The acid phosphatase values were adjustedto account for the percentage of phytase activity that appears in thepH2.5 acid phosphatase assay.) Nine of 425 transformants were found toover-produce both phytase and pH2.5 acid phosphatase; and, at greaterthan 4:1 HFU/PU (i.e., pH2.5 acid phosphatase HFU divided by phytasePU).

                  TABLE 13                                                        ______________________________________                                        DNA Constructs Used to Construct                                              Dual-Enzyme-Transformants of                                                  ALK0243 with Elevated Levels of                                               both pH 2.5 Acid Phosphatase and Phytase                                      Plasmid DNA Combination                                                                         Number Analyzed                                             ______________________________________                                        pPHO1/PFF4         3                                                          pPHO3/pFFI        108                                                         pPHO3/pFF2        50                                                          pPHO3/pFF4        11                                                          pPHO3(lin)/pFF6(lin)                                                                            74                                                          pPHO3(lin)/pFF8(lin)                                                                             7                                                          pPHO4A/pFF1       21                                                          pPHO4A/pFF2       22                                                          pPHO4A/pFF3       25                                                          pPHO4A/pFF4       22                                                          pFF6/prepho2       2                                                          pFIN-1A           84                                                          pFIN-1B           46                                                          Total             475                                                         ______________________________________                                    

Eighteen of the 475 combination transformants (Table 13) demonstratedactivities in excess of 20-fold HFU/ml greater than ALKO243 for pH2.5acid phosphatase and 250-fold PU/ml greater than ALKO243 for phytaseactivity (Table 14).

                                      TABLE 14                                    __________________________________________________________________________    A. niger ALK0243 Dual-Enzyme-Transformants Expressing Both                    Phytase and pH 2.5 Acid Phosphatase (pH 2.5AP) at Levels of Enzyme            Activity Greater than                                                         the ALK0243 parent cell by greater than 20-fold for pH 2.5 Acid               Phosphatase (A.P.) and                                                        greater than 250-fold for Phytase (Phy.).sup.a                                                    Activity                                                                          Activity                                                                          Ratio                                                                              Increase                                                                          Increase                                 Transformant                                                                         Plasmid(s)/Promoter AP/Phy                                                                 HFU/ml                                                                            PU/ml                                                                             HFU/PU                                                                             A.P.                                                                              Phytase                                  __________________________________________________________________________    GAX-11 pPHO-4A/pFF-4(GA/GA)                                                                       280,500                                                                           53,000                                                                            5.3   51 624                                      GAX-12 pPH0-4A/pFF-4(GA/GA)                                                                       176,000                                                                           29,000                                                                            6.1   32 342                                      GBE-14 pPH0-3/pFF-4 (GAPDH/GA)                                                                    147,300                                                                           29,400                                                                            5.0   27 346                                      GBH-134                                                                              pPH0-3/pFF-2 (GAPDH/β-T)                                                              291,500                                                                           39,000                                                                            7.5   53 459                                      GBH-157                                                                              pPH0-3/pFF-2 (GAPDH/β-T)                                                              247,500                                                                           54,000                                                                            4.6   45 635                                      GBJ-9  pFIN-JA (GA/GA)                                                                            212,400                                                                           38,800                                                                            5.5  39* 456                                      GBJ-10 pFIN-IA (GA/GA)                                                                            241,000                                                                           38,100                                                                            5.6  44* 445                                      GBJ-13 pFIN-JA (GA/GA)                                                                            259,800                                                                           25,300                                                                            10.2 47* 299                                      GBJ-16 pFIN-JA (GA/GA)                                                                            280,600                                                                           23,200                                                                            12.1 51* 273                                      GBJ-26 pFIN-JB (GA/GA)                                                                            328,100                                                                           45,700                                                                            7.2  60* 538                                      GBJ-27 pFIN-JB (GA/GA)                                                                            218,100                                                                           39,700                                                                            5.5  40* 467                                      GBJ-28 pFIN-1B(GA/GA)                                                                             372,300                                                                           23,600                                                                            15.8 68* 278                                      GBJ-31 pFIN-IB(GA/GA)                                                                             299,000                                                                           30,400                                                                            9.8  54* 355                                      GBJ-35 pFIN-IB(GA/GA)                                                                             255,300                                                                           28,400                                                                            9.0  46* 334                                      GBJ-35 pFIN-JB (GA/GA)                                                                            277,500                                                                           25,600                                                                            10.8 50* 301                                      GBJ-40 pFIN-4A (GA/GA)                                                                            401,000                                                                           38,200                                                                            10.5  73 449                                      GBJ-76 pPH0-4A/PFF-2 (GA/β-T)                                                                201,600                                                                           46,100                                                                            4.4  37* 542                                      GBJ-82 pPH0-4A/pFF-2 (GA/β-T)                                                                309,800                                                                           40,100                                                                            7.7  56* 472                                      ALK0243                                                                              control/native (native)                                                                     5,500                                                                               85                                                                             64.7  1   1                                       __________________________________________________________________________     .sup.a Enzyme activities were determined in shake flask cultures (as          described in Table 10, Example 4, above). The results are expressed as th     average of at least two independent fermentation cultures except (*) whic     was taken from a single fermentation culture. pH 2.5 acid phosphatase (pH     2.5 AP) levels were adjusted to subtract the background phytase activity      (i.e., the percentage of phytase that could be detected at pH 2.5 in the      pH 2.5 acid phosphatase assay conditions; Materials and Methods);             .sup.b All plasmids utilized in these studies were circular and in the        whole circular form. Plasmids pFIN1A and pFIN1B are single plasmids that      contain both the pH 2.5 acid phosphatase (A.P.) and phytase (Phy.) gene       sequence.                                                                     Transformant = Transformant designation; Promoter for acid phosphatase        (A.P)/promoter for physase (Phy));                                            GA = glucoamylase promoter;                                                   GAPDH = glyceraldehyde 3phosphate dehydrogenase promoter;                     T = Tubulin promoter;                                                         .sup.c HFU/ml, acid phosphatase units; PU/ml, phytase units (see Tables       7-10, above);                                                                 .sup.d Ratio HFU/PU = Activity for acid phosphatase (HFU/ml)/Activity for     phytase (PU/ml);                                                              .sup.e Increase is expressed as the fold increase in acid phosphatase         (HFU/ml) or phytase (PU/ml) activity in shake flask culture over the          activity with ALK0243 in shake flask culture (i.e., activity in HFU/ml or     PU/ml, respectively, of transformant per activity in HFU/ml or PU/ml,         respectively, of ALK0243).                                               

Molecular Characterization:

Strains GAX7, GAX-11 and GAX-12 were characterized to determine theirrelative amounts of gene dosage, message levels and enzyme production.The three transformants all show evidence of tandem integrationoccurring from the introduction of the phytase transformation vectorpFF4. Two of the strains, GAX-7 and GAX-11, also show evidence ofmultiple integration events (FIG. 17A). The increase in phytase messagelevel over ALKO243 mRNA levels varied from a 17.5-fold increase (forGAX-11) to a 34.4-fold increase (for GAX-12) (FIG. 17B; Table 15).

FIG. 17A shows Southern analysis of DNA copy number of phytase genes inthe dual-gene-transformed GAX strains. DNA fromdual-enzyme-transformants GAX-7, 11, and 12 was digested with BamHI andXbaI; electrophoresed; blotted; and, the filter was probed with a BamHI/XbaI fragment from pFF-4. Lane 1. pFF-4 plasmid control DNA digestedwith BamHI/XbaI. Lane 2. ALKO243 untransformed control DNA. Lane 3.Transformant GAX-7 DNA. Lane 4. Transformant GAX-11 DNA. Lane 5.Transformant GAX-12 DNA. p=transformation vector plasmid copy number;c=chromosomal gene copy number. FIG. 17B shows Northern analysis ofphytase mRNA transcript levels in the GAX strains of FIG. 17A: 20 mg oftotal RNA, spiked with 10 ng of mp 18 RF DNA (mp) was electrophoresed,blotted to nitrocellulose, and probed with pFF-1. The phytase transcriptis arrowed at 1.4 kb. Lane 1. ALKO243 untransformed control RNA. Lane 2.GAX-7 RNA. Lane 3. GAX-11 RNA. Lane 4. GAX-12 RNA.

                  TABLE 15                                                        ______________________________________                                        Comparison of Phytase Copy Number, mRNA Levels.                               and Enzyme Activity for Dual-Enzyme-Transformants.sup.a                                         Vector                                                      Strain Plasmid    Copy No..sup.b                                                                         mRNA.sup.c                                                                          Activity.sub.d                                                                       Increase.sup.e                        ______________________________________                                        ALK0243                                                                              Alone 1    .sup. 1.0                                                                               1.0     85   1                                    GAX-7  pFF4 (GA, cir)                                                                           2.sup.+  24.4  38,400 452                                   GAX-11 pFF4 (GA, cir)                                                                           6.sup.+  17.5  53,000 624                                   GAX-12 pFF4 (GA, cir)                                                                           8.sup.   34.4  29,000 341                                   ______________________________________                                         .sup.a GA = glucoamylase promoter; cir = circular vector;                     .sup.b Vector copy no. = the number of copies of the vector nucleic acid      in the genome of the trassformed cell; + = unquantitated additional copie     of phytase present;                                                           .sup.c mRNA = messenger RNA amplification levels, (as foldincrease over       levels in ALK0243);                                                           .sup.d Activity = phytase activity represented in PU/ml from shake flask      cultures as described in Table 10, above;                                     .sup.e Increase is expressed as the fold increase in phytase (PU/ml)          activity in shake flask culture over the activity with ALK0243 in shake       flask culture (i.e., activity in PU/ml, respectively, of transformant per     activity in PU/ml, respectively, of ALK0243).                            

Data from a similar analysis by Southern and Northern blotting of thethree strains are presented in Table 16, below, for pH2.5 acidphosphatase transformation vector gene sequences. The results show thatGAX-7, a pPHO-3 integrant, contained one extra copy of pH2.5 acidphosphatase (as a single integration event); whereas GAX-11 and 12,pPHO-4A integrants, contained extra copies (acquired by tandemintegration; FIG. 18A). Message levels for pH2.5 acid phosphatase wereincreased 2.7-fold (i.e., over levels in ALKO243) in GAX-7, and 10-foldand 15-fold in GAX-11 and GAX-12, respectively (FIG. 18B, Table 16). Asobserved for the single pH2.5 acid phosphatase gene transformants (i.e.,Example 4 above), higher copy numbers were obtained for integration oftransformation vector plasmids than with the phytase vectors, but themessage levels for pH2.5 acid phosphatase were not corresponding as highas the levels observed with the phytase vectors (Table 16, below).

FIG. 18A shows Southern analysis of DNA copy number of pH2.5 acidphosphatase sequences in GAX dual-gene-transformed strains. All sampleswere digested with SalI, electrophoresed, blotted to nitrocellulose, andprobed with the pH2.5 acid phosphatase gene as isolated in fragmentsfrom pAP-3 (Example 3, above). Lane 1. pPHO-3 plasmid DNA control, SalIdigested. Lane 2. pPHO-4A plasmid DNA control, SalI digested. Lane 3.Untransformed ALKO243 DNA control. Lane 4. GAX-7 DNA. Lane 5. GAX-11DNA. Lane 6. GAX-12 DNA.

FIG. 18B shows Northern analysis of mRNA levels of pH2.5 acidphosphatase message expression in dual-enzyme transformed in strains.The pH2.5 acid phosphatase RNA transcript is arrowed. Lane 1. ALKO243untransformed control RNA. Lane 2. GAX-7 RNA. Lane 3. RNA. GAX-11 RNA.Lane 4. GAX-12 RNA.

                  TABLE 16                                                        ______________________________________                                        Comparison of pH 2.5 acid phosphatase copy number, mRNA Levels                and Enzyme Activity in Dual-Enzyme-Transformants.sup.a                                            Vector         Ac-   In-                                  Strain Plasmid      Copy No..sup.b                                                                         mRNA.sup.c                                                                          tivity.sup.d                                                                        crease.sup.e                         ______________________________________                                        ALK0243                                                                              --             1.0    1      5,500                                                                                1.0                                GAX-7  pPH03 (GAP, cir)                                                                            1        2.7  112,100                                                                             20                                   GAX-11 pPH04A (GA, cir)                                                                           16       10.3  280,500                                                                             51                                   GAX-12 pPH04A (GA, cir)                                                                           16       16.0  176,000                                                                             32                                   ______________________________________                                         .sup.a GAP = glyceraldehyde 3phosphate dehydrogenase promoter; GA =           glucoamylase promoter; cir = circular vector;                                 .sup.b Vector copy no., see Table 15, above;                                  .sup.c mRNA = messenger RNA amplification levels, (as foldincrease over       Levels in ALK0243);                                                           .sup.d Activity = pH 2.5 acid phosphatase activity represented in HFU/ml      measured in shake flask cultures as described in Table 10, above;             .sup.e Increase is expressed as the fold increase in acid phosphatase         (HFU/ml) activity in shake flask culture over the activity with ALK0243 i     shake flask culture (i.e., activity in HFU/ml of transformant per activit     in HFU/ml of ALK0243).                                                   

Materials and Methods:

The materials and methods used herein were the same as those used above(see Example 4).

EXAMPLE 6 Control Of Over-Expression Levels Of Phytase And pH2.5 AcidPhosphatase In Dual-Transformants

As described above in Examples 2-5, industrially significant increasesin the yields of phytase and pH2.5 acid phosphatase were achieved bytransforming a strain of Aspergillus niger (i.e., ALKO2268) withtransformation vector constructs and selecting for transformants thatover-expressed the desired enzymatically active protein. In shake flaskfermentations, phytase yields were increased 2,000-fold over enzymeproduction levels achieved with ALKO243 (Example 4; Table 8, above), andpH2.5 acid phosphatase yields were increased more than 100-fold (Example4; Table 11, above). These yield increases were considered to resultfrom several factors: most importantly, increased gene dosage, andutilization of effective alternative promoters (i.e., heterologouspromoters GA, GAP, GAPDH and the β-tubulin promoter; see Examples 4 and5, above). Factors controlling expression levels were evaluated further.

The effect of gene dosage on fermentation levels of phytase and acidphosphatase:

The data presented in the Examples 4 and 5 above suggest that relativelylarge increases in phytase protein resulted from relatively modestincreases in gene copy number and mRNA transcript levels. The observedgene copy number increases ranged from three to five additional copiesin the phytase over-producing strains. These strains, namely GAE-32,GAK-47, GAL-65, GAM-225 and GAO-248, apparently acquired the additionalgene copies by multiple integration events of the recombinanttransformation vector plasmid DNA, i.e., rather than by tandemintegration. Three of the five strains, GAL-65, GAM-225 and GAO-268,were transformed with linear DNA fragments, from which tandem arrays maybe less likely to occur. GAE-32 and GAK-47, however, were transformedwith circular vector constructions, which often give rise to tandemlyarranged gene copies. Since the transformants were selected on the basisof phytase activity in the enzyme-plate assay (rather than by Southernblot analysis, as is commonly the case), the relative frequency oftransformants that had integrated phytase DNA sequences in optimallocations was likely increased. Chromosomal integration site(s)apparently play an important role in phytase gene expression and thisscreening method selected optimized transformants.

In high producing phytase transformants, messenger RNA levels wereelevated seven- to thirteen-fold over ALKO2268, while fermentationlevels produced by the cells increased fifty-fold.

The copy number observed in pH2.5 acid phosphatase transformants (Table12, Example 4; Table 16, Example 5, above) was higher than in phytasetransformants (Table 9, Example 4; Table 15, Example 5, above), but theamounts of pH2.5 acid phosphatase message was substantially lower thanin phytase transformants. High pH2.5 acid phosphatase over-producerstrains GAT-143, GAW-54, GAW-89, GAW-121 and GAW-130 were transformedwith vectors having linear DNA from either pPHO-3 or pPHO-4A. Seven totwelve additional gene copies (i.e., vector gene copies) were seen inthese transformants, but message levels were only increased by aboutthree- to about seven-fold.

Effects of linear and circular DNA on titers of transformants:

The effects of linear DNA transformation vector constructs and circularvector constructs on phytase enzyme production were evaluated. Overall,circular constructions gave a higher frequency of transformants withincreased phytase enzyme production (FIG. 19), but the maximal levels ofenzyme produced by the transformants were comparable with linear andcircular constructs. For example, circular vector pFF3 gave 8/24 (33%)phytase transformants with enzyme production levels greater than 750 PU.The linear counterpart of pFF3, i.e., pFF8, had two-fold fewertransformants (i.e., that achieved the same threshold level of phytaseproduction), but the transformants that were selected had maximalproduction levels comparable with pFF8 transformants. When linearfragments were purified and religated, the frequency of higher producersdid not increase.

FIG. 19. Comparison of the effects of linear and circular DNA on thetiters of twenty-four randomly selected phytase transformants. With thecircular vector pFF3, 33% of the phytase transformants had levels ofenzyme production greater than 750 PNU. Its linear counterpart, pFF8,had two-fold fewer transformants that achieved that same threshold ofenzyme production. PNU=phytase normalized units.

Effects of fungal promoters on product titers:

The level of expression of genes in Aspergillus may be dependent uponthe particular choice of A. niger filamentous fungal promoter. Theresults presented in Examples 4 and 5, above, support the notion thatphytase and pH2.5 acid phosphatase can be expressed from avariety of A.niger promoters (e.g., including the GA, GAPDH and β-tubulin promoter),as well as the native promoter elements in the 5' regulatory regions ofthe phytase and pH2.5 acid phosphatase genes. However, the results alsoshow that the maximal levels of enzyme produced in transformants, fromexpression directed by the different promoters, is different; i.e., thechoice of promoter determined the level of expression. While the nativephytase and pH2.5 acid phosphatase promoters were effective, theglucoamylase (GA) and GAPDH promoters were significantly better.Production levels of phytase and pH2.5 acid phosphatase in fermentationculture under the control of the GA and GAPDH promoters are shown inFIG. 20.

FIG. 20. Different fungal promoters used in transforming plasmidsinfluenced the level of phytase and pH2.5 acid phosphatase produced byA. niger transformants. The levels of enzyme produced by tentransformants (i.e., with the highest levels for that given promoter),were averaged together. (Data from dual-enzyme-transformants were notused in these calculations.) GAPDH=glyceraldehyde 3-phosphatedehydrogenase. GA=glucoamylase.

The glucoamylase promoter from Aspergillus species has been reportedlyused to increase expression of several proteins (Ullah et al.,1987). Inaddition to the GA promoter, the GA sequence contains a glucoamylasesignal peptide and propeptide that have been reportedly used in attemptsto increase expression (Yamamoto et al., 1970).

To test the effects of the phytase signal sequence on the levels ofphytase produced, the signal sequence in the phytase gene was replaced(in construct pFF-11) by a synthetic oligonucleotide containing thefungal glucoamylase putative signal sequence. Surprisingly, instead ofincreasing the levels of enzyme produced, the insertion of the GA signalsequence actually decreased the levels of phytase produced, as comparedwith the levels of production achieved in transformants having thenative phytase signal sequence (i.e., in the native phytase genesequence). These results indicate the possible importance of phytasesignal sequence in maximizing the levels of production of phytase.

Genes under the control of a glucoamylase promoter may be upregulated(induced) in the presence of starch. To evaluate the effects of suchupregulation on levels of phytase production, some pFF-4 and pFF-9glucoamylase-promoter phytase transformants were cultured in media thathad been supplemented with 4% corn starch. Levels of enzyme productionwere increased an average of 1.4-fold in the latter cultures, althoughthese levels of production were not consistently observed. The highestyields of phytase seen under inducing conditions were 3240-3770PNU. Theresults indicate that optimization of the media used in the phytaseproduction fermentation broth could result in significantly greateryields of enzyme.

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter isreportedly a constitutive promoter that may drive high-level expressionof a heterologous gene product. The inventors independently cloned theglyceraldehyde phosphate dehydrogenase A gene (gpdA) from A. nigerATCCI1015 in order to evaluate its effect on phytase and pH2.5 acidphosphatase gene expression in a transformation vector constructintroduced into A. niger strain ALKO243 or ALKO2268. The levels ofenzyme production in transformants having the GAPDH promoter drivingexpression of phytase and pH2.5 acid phosphatase were nearly identicalto the levels achieved, above, using the glucoamylase promoter (FIG.20). Thus, the GAPDH promoter did not appear to offer any uniqueopportunities for increased enzyme production. The reasons for thisparticular behavior are presently unclear; however, such variability inexpression between ALKO243 and ALKO2268 may relate to the derivation ofALKO2268 as a mutant from ALKO243, and in this regard ALKO2268 producesonly about one-half of the amount of pH2.5 acid phosphatase produced bythe parental ALKO243 strain. Thus, it is possible that ALKO2268 isdeficient in some limiting factors relating to expression of pH2.5 acidphosphatase. (Such strain-dependent differences in promoter-drivenexpression were not observed with any other promoters.)

The β-tubulin promoter is also reportedly a promoter capable of drivingconstitutive expression of genes. To tests its possible effects onphytase and pH2.5 acid phosphatase gene expression, the β-tubulinpromoter was isolated from A. niger strain 1015. The promoter wasintroduced into transformation vectors to evaluate its effects on thelevels of phytase and pH2.5 acid phosphatase production. Previousstudies by the inventors suggested that expression of homologous fungalgenes could be increased when driven by the β-tubulin promoter, and thatthis increase was substantially greater than that which could beachieved through the use of a GAPDH- or GA-promoter (Panlabs,unpublished) Unfortunately, inclusion of a β-tubulin promoter in thephytase transformation vector construct actually decreased the levels ofenzyme produced; and, with pH2.5 acid phosphatase only a modest increasein production levels was noted over the levels of production mediated bythe native promoter (FIG. 20).

It was also considered possible that production of recombinant phytaseand pH2.5 acid phosphatase might be sensitive to phosphate-mediatedrepression of transcription. In this case, the levels of enzyme producedmight be increased by eliminating such conditions in the fermentationculture. In support of the notion of phosphate-mediated repression,cells of both the ALKO243 and ALKO2268 strains produced no detectablephytase when excess phosphate was added to the fermentation culturebroth. To study this possibility further, the levels of enzyme producedby phytase transformants having alternative promoters (i.e., GA, GAPDHor β-tubulin) were compared to the levels achieved with the nativephytase and pH2.5 acid phosphatase promoters in the transformationvector constructs to see if the use of such an alternative promotereliminated the putative phosphate-mediated repression. Interestingly, nophytase production was detected in either the phytase or the pH2.5 acidphosphatase promoter when the transformants were grown in the presenceof phosphate, supporting the notion of phosphate-mediated repressionthat cannot be overcome through use of the native promoter integratedinto a different chromosomal site. In evaluating the alternativepromoters, one transformant with a β-tubulin promoter was able toproduce phytase in the presence of phosphate, but it showed a decreasein phytase production by about one-half In contrast, GAPDH-promoter andGA-promoter transformants were not sensitive to phosphate-mediatedinhibition to any degree, and yielded similar levels of phytaseproduction in the presence or absence of additional phosphate. Thus, theresults suggest that regulatory elements in the 5' regulatory region ofthe phytase and pH2.5 acid phosphatase genes may be useful for directingphosphate-mediated repression of gene expression, and that insertion ofalternative promoters into the 5' regions of these genes can overcomethese native regulatoryconstraints and increase production yields.

The identification of rate limiting factors, whether energy or secretionrelated, may further increase yields of phytase and pH2.5 acidphosphatase. Other factors such as media optimization, increased orstabilized gene transcription levels of pH2.5 acid phosphatase, orclassical mutagenesis screening of high producers could have additionalimpacts on product yield. Elevated gene dosage and the use ofalternative promoters have dramatically increased production yields overnative promoters and escaped negative regulatory constraints. Thecloning and reintroduction of the genes for phytase and pH2.5 acidphosphatase has resulted in commercially significant levels of theseenzymes for use as animal feed supplements. Rational design ofhomologous gene over-expression can be applied to increase the yields ofother fungal industrial products, and potentially give a betterunderstanding of the mechanisms required for heterologous expression.

Materials and Methods:

The materials and methods used in this example are the same as thoseused in Example 4, above.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

CITATIONS

Arima, K. et al., "The nucleotide sequence of the yeast PH05 gene: aputative precursor of repressible acid phosphatase contains a signalpeptide." Nucleic Acids Res. 11:1657-1672, 1983.

Bajwa, W. et al., "Structural analysis of the two tandemly repeated acidphosphatase genes in yeast," Nucleic Acids Res. 11:7721-7739, 1983.

Elliot, S. et al., "Isolation and characterization of the structuralgene for secreted acid phosphatase from Schizosaccharomyces pombe," J.Biol. Chem. 261:2936-2941, 1986.

Touati, E. et al., "The structure of the promoter and amino terminalregion of pH2.5 acid phosphatase structural gene (appA) of E. coli: anegative control of transcription mediated by cyclic AMP, " Biochemie69:215-221, 1987.

Han, Y. W., et al., "Phosphatase production by Aspergillus ficuum,"Journal of Industrial Microbiology 1:295-301, 1987.

Himeno, M. et al., "Isolation and Sequencing of a cDNA Clone EncodingAcid Phosphatase in Rat Liver Lysosomes," Biochem. Biophys. Res. Comm.162:1044-1053, 1989.

Kalkknen, N. et al. "A Gas-Pulsed-Liquid-Phase Sequencer ConstructedFrom a Beckman 8900 By Using Applied Biosystems Delivery and CartridgeBlocks," J. Prot. Chem. 7:242-243, 1988.

Lloyd, A. T. et al., "Codon usage in Aspergillus nidulans," Mol GenGenet 230:288-294, 1991.

McAda, P. C. et al., "A Yeast Mitochondrial Chelator-Sensitive ProteaseThat Processes Cytoplasmically Synthesized Protein Precursors,"Isolation from Yeast and Assay in Methods in Enzymology 97:337-344,1983.

Needleman, S. B. et al., "General Method Applicable to the Search forSimilarities in the Amino Acid Sequence of Two Proteins ," J. Mol. Biol.48:443-453, 1970.

Pohlmann, R. et al., EMBO Journal 7:2343-2350, 1988.

Rambosek, J. et al., "Recombinant DNA in Filamentous Fungi: Progress andProspects," CRC Critical Reviews in Biotechnology 6:357-393, 1987.

Roiko, L. et al., Gene 89:223-229, 1990.

Sanger et al., Proc. Nat. Acad. Sci. USA 74:5463-5467, 1977.

Shieh, T. R. et al., "Regulation of the Formation of Acid Phosphatase byInorganic Phosphate in Aspergillus ficuum," Journal of Bacteriology100:1161-1165, 1969.

Shieh, T. R. et al., "Survey of Microorganisms for the Production ofExtracellular Phytase," Appl Microbiol 6:1348-1351, 1968.

Standen, R., Nucleic Acids Res. 12:505-519, 1984.

Tailor, P. et al., Nucleic Acids Research 18:4921-4928, 1990.

Tilburn, J. et al., Gene 26:205-221, 1983.

Ullah, H. J. et al., "Cyclohexanedione Modification Of Arginine at theActive Site of Aspergillus ficuum Phytase," Biochemical and BiophysicalResearch Communications 178(1):45-53, 1991.

Ullah, H. J., "Aspergillus ficuum Phytase: Partial Primary Structure,Substrate Selectivity, and Kinetic Characterization," PreparativeBiochemistry 18(4):459-471, 1988.

Ullah, H. J., et al., "Purification, N-terminal Amino Acid Sequence andCharacterization of pH2.5 optimum acid phosphatase (E.C. 3.1.3.2) fromAspergillus ficuum," Preparative Biochemistry, 17(4):397-422, 1987.

Yamamoto, K. R. et al., "Rapid Bacteriophage Sedimentation in thePresence of Polyethylene Glycol and its Application to Large-Scale VirusPurification," Virology 40:734-744, 1970.

Shieh and Ware, U.S. Patent No. 3,297,548.

Nelson, T. S. et al., J. Nutrition 101:1289-1294, 1971.

Sandberg, A-S. et al., Journal of Nutrition 117:2061-2065, 1987.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 94                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2379 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(404..447, 550..1906)                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGATGGCGGCCTAGGGCATCCAGGCACCCTTTCCCAACGGGGGAACTTCCGTTGTCCACG60                TGCCCTGGTTCAGCCAATCAAAGCGTCCCACGGCAATGCTGGATCAACGATCAACTTGAA120               TGCAATAAATGAAGATGCAACTAACACCATCTGTTGCCTTTCTCTCGAGAAAGCTCCTCC180               ACTTCTCACACTAGATTTATCCGTTCCTTGTCGACTTCCCGTCCCATTCGGCCTCGTCCA240               CTGAAGATCTATCCCACCATTGCACGTGGGCCACCTTTGTGAGCTTCTAACCTGAACTGG300               TAGAGTATCACACAACATGCGAAAGTGGGATGAAGGGGTTATATGAGGACCGTCCGGTCC360               GGCGCGATGGCCGTAGCTGCCAATCGCTGCTGTGCAAGAAATTTCTTCTCATAGGCATC419                ATGGGCGTCTCTGCTGTTCTACTTCCTTTGTATCTCCTAGCTGGGTATGCTA471                       MetGlyValSerAlaValLeuLeuProLeuTyrLeuLeuAlaGly                                 151015                                                                        AGCACCGCTATCTAAGTCTGATAAGGACCCTCTTTGCCGAGGGCCCCTGAAGCTCGGACT531               GTGTGGGACTACTGATCGCTGACAATCTGTGCAGAGTCACCTCCGGACTGGCA584                      ValThrSerGlyLeuAla                                                            20                                                                            GTCCCCGCCTCGAGAAATCAATCCACTTGCGATACGGTCGATCAAGGG632                           ValProAlaSerArgAsnGlnSerThrCysAspThrValAspGlnGly                              253035                                                                        TATCAATGCTTCTCCGAGACTTCGCATCTTTGGGGTCAATACGCGCCG680                           TyrGlnCysPheSerGluThrSerHisLeuTrpGlyGlnTyrAlaPro                              404550                                                                        TTCTTCTCTCTGGCAAACGAATCGGCCATCTCCCCTGATGTGCCCGCC728                           PhePheSerLeuAlaAsnGluSerAlaIleSerProAspValProAla                              556065                                                                        GGTTGCAGAGTCACTTTCGCTCAGGTCCTCTCCCGTCATGGAGCGCGG776                           GlyCysArgValThrPheAlaGlnValLeuSerArgHisGlyAlaArg                              70758085                                                                      TATCCGACCGAGTCCAAGGGCAAGAAATACTCCGCTCTCATTGAGGAG824                           TyrProThrGluSerLysGlyLysLysTyrSerAlaLeuIleGluGlu                              9095100                                                                       ATCCAGCAGAACGTGACCACCTTTGATGGAAAATATGCCTTCCTGAAG872                           IleGlnGlnAsnValThrThrPheAspGlyLysTyrAlaPheLeuLys                              105110115                                                                     ACATACAACTACAGCTTGGGTGCAGATGACCTGACTCCCTTCGGAGAG920                           ThrTyrAsnTyrSerLeuGlyAlaAspAspLeuThrProPheGlyGlu                              120125130                                                                     CAGGAGCTAGTCAACTCCGGCATCAAGTTCTACCAGCGATACGAATCG968                           GlnGluLeuValAsnSerGlyIleLysPheTyrGlnArgTyrGluSer                              135140145                                                                     CTCACAAGGAACATCATTCCGTTCATCCGATCCTCTGGCTCCAGCCGC1016                          LeuThrArgAsnIleIleProPheIleArgSerSerGlySerSerArg                              150155160165                                                                  GTGATCGCCTCCGGCGAGAAATTCATTGAGGGCTTCCAGAGCACCAAG1064                          ValIleAlaSerGlyGluLysPheIleGluGlyPheGlnSerThrLys                              170175180                                                                     CTGAAGGATCCTCGTGCCCAGCCGGGCCAATCGTCGCCCAAGATCGAC1112                          LeuLysAspProArgAlaGlnProGlyGlnSerSerProLysIleAsp                              185190195                                                                     GTGGTCATTTCCGAGGCCAGCTCATCCAACAACACTCTCGACCCAGGC1160                          ValValIleSerGluAlaSerSerSerAsnAsnThrLeuAspProGly                              200205210                                                                     ACCTGCACTGTCTTTGAAGACAGCGAATTGGCCGATACCGTCGAAGCC1208                          ThrCysThrValPheGluAspSerGluLeuAlaAspThrValGluAla                              215220225                                                                     AATTTCACCGCCACGTTCGCCCCCTCCATTCGTCAACGTCTGGAGAAC1256                          AsnPheThrAlaThrPheAlaProSerIleArgGlnArgLeuGluAsn                              230235240245                                                                  GACCTGTCTGGCGTGACTCTCACAGACACAGAAGTGACCTACCTCATG1304                          AspLeuSerGlyValThrLeuThrAspThrGluValThrTyrLeuMet                              250255260                                                                     GACATGTGCTCCTTCGACACCATCTCCACCAGCACCGTCGACACCAAG1352                          AspMetCysSerPheAspThrIleSerThrSerThrValAspThrLys                              265270275                                                                     CTGTCCCCCTTCTGTGACCTGTTCACCCATGACGAATGGATCCACTAC1400                          LeuSerProPheCysAspLeuPheThrHisAspGluTrpIleHisTyr                              280285290                                                                     GACTACCTCCAGTCCCTGAAAAAATACTACGGCCATGGCGCAGGTAAC1448                          AspTyrLeuGlnSerLeuLysLysTyrTyrGlyHisGlyAlaGlyAsn                              295300305                                                                     CCGCTCGGCCCGACCCAGGGCGTCGGCTACGCTAACGAGCTCATCGCC1496                          ProLeuGlyProThrGlnGlyValGlyTyrAlaAsnGluLeuIleAla                              310315320325                                                                  CGTCTCACCCACTCGCCTGTCCACGATGACACCAGCTCCAACCACACC1544                          ArgLeuThrHisSerProValHisAspAspThrSerSerAsnHisThr                              330335340                                                                     TTGGACTCGAACCCAGCTACCTTCCCGCTCAACTCTACTCTCTACGCG1592                          LeuAspSerAsnProAlaThrPheProLeuAsnSerThrLeuTyrAla                              345350355                                                                     GACTTTTCCCACGATAACGGCATCATCTCTATCCTCTTTGCTTTGGGT1640                          AspPheSerHisAspAsnGlyIleIleSerIleLeuPheAlaLeuGly                              360365370                                                                     CTGTACAACGGCACTAAGCCGCTGTCTACCACGACCGTGGAGAATATC1688                          LeuTyrAsnGlyThrLysProLeuSerThrThrThrValGluAsnIle                              375380385                                                                     ACCCAGACAGATGGGTTCTCGTCTGCTTGGACGGTTCCGTTTGCTTCG1736                          ThrGlnThrAspGlyPheSerSerAlaTrpThrValProPheAlaSer                              390395400405                                                                  CGTCTGTACGTCGAGATGATGCAGTGCCAGGCCGAGCAGGAGCCGCTG1784                          ArgLeuTyrValGluMetMetGlnCysGlnAlaGluGlnGluProLeu                              410415420                                                                     GTCCGTGTCTTGGTTAATGATCGCGTTGTCCCGCTGCATGGGTGTCCA1832                          ValArgValLeuValAsnAspArgValValProLeuHisGlyCysPro                              425430435                                                                     ATTGATGCTTTGGGGAGATGTACCCGGGATAGCTTTGTGAGGGGGTTG1880                          IleAspAlaLeuGlyArgCysThrArgAspSerPheValArgGlyLeu                              440445450                                                                     AGCTTTGCTAGATCTGGGGGTGATTGGGCGGAGTGTTCTGCT1922                                SerPheAlaArgSerGlyGlyAspTrpAlaGluCysSerAla                                    455460465                                                                     TAGCTGAACTACCTTGATGGATGGTATGTATCAATCAGAGTACATATCATTACTTCATGT1982              ATGTATTTACGAAGATGTACATATCGAAATATCGATGATGACTACTCCGGTAGATATTTG2042              GTCCCCTTCTATCCTTCGTTCCACAACCATCGCACTCGACGTACAGCATAATACAACTTC2102              AGCATTAACAAACGAACAAATAATATTATACACTCCTCCCCAATGCAATAACAACCGCAA2162              TTCATACCTCATATAGATACAATACAATACATCCATCCCTACCCTCAAGTCCACCCATCC2222              CATAATCAAATCCCTACTTACTCCTCCCCCTTCCCAGAACCCACCCCCGAAGGAGTAATA2282              GTAGTAGTAGAAGAAGCAGACGACCTCTCCACCAACCTCTTCGGCCTCTTATCCCCATAC2342              GCTATACACACACGAACACACCAAATAGTCAGCATGC2379                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 467 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyValSerAlaValLeuLeuProLeuTyrLeuLeuAlaGlyVal                              151015                                                                        ThrSerGlyLeuAlaValProAlaSerArgAsnGlnSerThrCysAsp                              202530                                                                        ThrValAspGlnGlyTyrGlnCysPheSerGluThrSerHisLeuTrp                              354045                                                                        GlyGlnTyrAlaProPhePheSerLeuAlaAsnGluSerAlaIleSer                              505560                                                                        ProAspValProAlaGlyCysArgValThrPheAlaGlnValLeuSer                              65707580                                                                      ArgHisGlyAlaArgTyrProThrGluSerLysGlyLysLysTyrSer                              859095                                                                        AlaLeuIleGluGluIleGlnGlnAsnValThrThrPheAspGlyLys                              100105110                                                                     TyrAlaPheLeuLysThrTyrAsnTyrSerLeuGlyAlaAspAspLeu                              115120125                                                                     ThrProPheGlyGluGlnGluLeuValAsnSerGlyIleLysPheTyr                              130135140                                                                     GlnArgTyrGluSerLeuThrArgAsnIleIleProPheIleArgSer                              145150155160                                                                  SerGlySerSerArgValIleAlaSerGlyGluLysPheIleGluGly                              165170175                                                                     PheGlnSerThrLysLeuLysAspProArgAlaGlnProGlyGlnSer                              180185190                                                                     SerProLysIleAspValValIleSerGluAlaSerSerSerAsnAsn                              195200205                                                                     ThrLeuAspProGlyThrCysThrValPheGluAspSerGluLeuAla                              210215220                                                                     AspThrValGluAlaAsnPheThrAlaThrPheAlaProSerIleArg                              225230235240                                                                  GlnArgLeuGluAsnAspLeuSerGlyValThrLeuThrAspThrGlu                              245250255                                                                     ValThrTyrLeuMetAspMetCysSerPheAspThrIleSerThrSer                              260265270                                                                     ThrValAspThrLysLeuSerProPheCysAspLeuPheThrHisAsp                              275280285                                                                     GluTrpIleHisTyrAspTyrLeuGlnSerLeuLysLysTyrTyrGly                              290295300                                                                     HisGlyAlaGlyAsnProLeuGlyProThrGlnGlyValGlyTyrAla                              305310315320                                                                  AsnGluLeuIleAlaArgLeuThrHisSerProValHisAspAspThr                              325330335                                                                     SerSerAsnHisThrLeuAspSerAsnProAlaThrPheProLeuAsn                              340345350                                                                     SerThrLeuTyrAlaAspPheSerHisAspAsnGlyIleIleSerIle                              355360365                                                                     LeuPheAlaLeuGlyLeuTyrAsnGlyThrLysProLeuSerThrThr                              370375380                                                                     ThrValGluAsnIleThrGlnThrAspGlyPheSerSerAlaTrpThr                              385390395400                                                                  ValProPheAlaSerArgLeuTyrValGluMetMetGlnCysGlnAla                              405410415                                                                     GluGlnGluProLeuValArgValLeuValAsnAspArgValValPro                              420425430                                                                     LeuHisGlyCysProIleAspAlaLeuGlyArgCysThrArgAspSer                              435440445                                                                     PheValArgGlyLeuSerPheAlaArgSerGlyGlyAspTrpAlaGlu                              450455460                                                                     CysSerAla                                                                     465                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2071 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(136..915, 970..1089, 1142..1245,                           1305..1737)                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCATGCTGGACCGCAATCTCCGATCGCCGGGTATAAAAGGTCCTCCAAACCCCTCTCGGT60                CGATATGTACCCCGCTCGTCATCTCCAATCCTCTCGAGAGCACCTTCTCCAGCTTTTGTC120               AATTGTACCTTCGCAATGCCTCGCACCTCTCTCCTCACCCTGGCCTGTGCT171                        MetProArgThrSerLeuLeuThrLeuAlaCysAla                                          1510                                                                          CTGGCCACGGGCGCATCCGCTTTCTCCTACGGCGCTGCCATTCCTCAG219                           LeuAlaThrGlyAlaSerAlaPheSerTyrGlyAlaAlaIleProGln                              152025                                                                        TCAACCCAGGAGAAGCAGTTCTCTCAGGAGTTCCGCGATGGCTACAGC267                           SerThrGlnGluLysGlnPheSerGlnGluPheArgAspGlyTyrSer                              303540                                                                        ATCCTCAAGCACTACGGTGGTAACGGACCCTACTCCGAGCGTGTGTCC315                           IleLeuLysHisTyrGlyGlyAsnGlyProTyrSerGluArgValSer                              45505560                                                                      TACGGTATCGCTCGCGATCCCCCGACCAGCTGCGAGGTCGATCAGGTC363                           TyrGlyIleAlaArgAspProProThrSerCysGluValAspGlnVal                              657075                                                                        ATCATGGTCAAGCGTCACGGAGAGCGCTACCCGTCCCCTTCAGCCGGC411                           IleMetValLysArgHisGlyGluArgTyrProSerProSerAlaGly                              808590                                                                        AAGGACATCGAAGAGGCCCTGGCCAAGGTCTACAGCATCAACACTACT459                           LysAspIleGluGluAlaLeuAlaLysValTyrSerIleAsnThrThr                              95100105                                                                      GAATACAAGGGCGACCTGGCCTTCCTGAACGACTGGACCTACTACGTC507                           GluTyrLysGlyAspLeuAlaPheLeuAsnAspTrpThrTyrTyrVal                              110115120                                                                     CCTAATGAGTGCTACTACAACGCCGAGACCACCAGCGGCCCCTACGCC555                           ProAsnGluCysTyrTyrAsnAlaGluThrThrSerGlyProTyrAla                              125130135140                                                                  GGTTTGCTGGACGCGTACAACCATGGCAACGATTACAAGGCTCGCTAC603                           GlyLeuLeuAspAlaTyrAsnHisGlyAsnAspTyrLysAlaArgTyr                              145150155                                                                     GGCCACCTCTGGAACGGTGAGACGGTCGTGCCCTTCTTTTCTAGTGGC651                           GlyHisLeuTrpAsnGlyGluThrValValProPhePheSerSerGly                              160165170                                                                     TACGGACGTGTCATCGAGACGGCCCGCAAGTTCGGTGAGGGTTTCTTT699                           TyrGlyArgValIleGluThrAlaArgLysPheGlyGluGlyPhePhe                              175180185                                                                     GGCTACAACTACTCCACCAACGCTGCCCTCAACATCATCTCCGAGTCC747                           GlyTyrAsnTyrSerThrAsnAlaAlaLeuAsnIleIleSerGluSer                              190195200                                                                     GAGGTCATGGGCGCGGACAGCCTCACGCCCACCTGTGACACCGACAAC795                           GluValMetGlyAlaAspSerLeuThrProThrCysAspThrAspAsn                              205210215220                                                                  GACCAGACCACCTGCGACAACCTGACTTACCAGCTGCCCCAGTTCAAG843                           AspGlnThrThrCysAspAsnLeuThrTyrGlnLeuProGlnPheLys                              225230235                                                                     GTCGCTGCTGCCCGCCTAAACTCCCAGAACCCCGGCATGAACCTCACC891                           ValAlaAlaAlaArgLeuAsnSerGlnAsnProGlyMetAsnLeuThr                              240245250                                                                     GCATCTGATGTCTACAACCTGATGGGTATGTGATTACGGTACAATCATTGGCTC945                     AlaSerAspValTyrAsnLeuMet                                                      255260                                                                        AAACCTCCAGCTGACAGCATCCTAGTTATGGCCTCCTTTGAGCTCAATGCT996                        ValMetAlaSerPheGluLeuAsnAla                                                   265                                                                           CGTCCCTTCTCCAACTGGATCAACGCCTTTACCCAGGACGAATGGGTC1044                          ArgProPheSerAsnTrpIleAsnAlaPheThrGlnAspGluTrpVal                              270275280285                                                                  AGCTTCGGTTACGTTGAGGATTTGAACTACTACTACTGCGCTGGG1089                             SerPheGlyTyrValGluAspLeuAsnTyrTyrTyrCysAlaGly                                 290295300                                                                     TGAGTTTACCATTTGATCCATTATTGTCTTGGATCAGCTAACGATCGATAGTCCC1144                   Pro                                                                           GGTGACAAGAACATGGCTGCTGTGGGTGCCGTCTACGCCAACGCCAGT1192                          GlyAspLysAsnMetAlaAlaValGlyAlaValTyrAlaAsnAlaSer                              305310315                                                                     CTCACCCTCCTGAACCAGGGACCCAAGGAAGCCGGCTCCTTGTTCTTC1240                          LeuThrLeuLeuAsnGlnGlyProLysGluAlaGlySerLeuPhePhe                              320325330                                                                     AACTTGTACGTTCTCGGCAGAATCAGAGTCTCACAAAAAGAAACTCTTCACTAACA1296                  AsnPhe                                                                        335                                                                           TATAGTAGTGCCCACGACACCAACATCACCCCCATCCTCGCCGCCCTA1344                          AlaHisAspThrAsnIleThrProIleLeuAlaAlaLeu                                       340345                                                                        GGCGTCCTCATCCCCAACGAGGACCTTCCTCTTGACCGGGTCGCCTTC1392                          GlyValLeuIleProAsnGluAspLeuProLeuAspArgValAlaPhe                              350355360                                                                     GGCAACCCCTACTCGATCGGCAACATCGTGCCCATGGGTGGCCATCTG1440                          GlyAsnProTyrSerIleGlyAsnIleValProMetGlyGlyHisLeu                              365370375380                                                                  ACCATCGAGCGTCTCAGCTGCCAGGCCACCGCCCTCTCGGACGAGGGT1488                          ThrIleGluArgLeuSerCysGlnAlaThrAlaLeuSerAspGluGly                              385390395                                                                     ACCTACGTGCGTCTGGTGCTGAACGAGGCTGTACTCCCCTTCAACGAC1536                          ThrTyrValArgLeuValLeuAsnGluAlaValLeuProPheAsnAsp                              400405410                                                                     TGCACCTCCGGACCGGGCTACTCCTGCCCTCTGGCCAACTACACCTCC1584                          CysThrSerGlyProGlyTyrSerCysProLeuAlaAsnTyrThrSer                              415420425                                                                     ATCCTGAACAAGAATCTGCCAGACTACACGACCACCTGCAATGTCTCT1632                          IleLeuAsnLysAsnLeuProAspTyrThrThrThrCysAsnValSer                              430435440                                                                     GCGTCCTACCCGCAGTATCTGAGCTTCTGGTGGAACTACAACACCACG1680                          AlaSerTyrProGlnTyrLeuSerPheTrpTrpAsnTyrAsnThrThr                              445450455460                                                                  ACGGAGCTGAACTACCGCTCTAGCCCTATTGCCTGCCAGGAGGGTGAT1728                          ThrGluLeuAsnTyrArgSerSerProIleAlaCysGlnGluGlyAsp                              465470475                                                                     GCTATGGACTAGATGCAGAGGGGTAGGTCCCGGGATACTTTAGTGATGA1777                         AlaMetAsp                                                                     TTGATATTCAAGTTTGGTGGTGACGATCACCTTGTTAATAGTCTTGTACAGTCATACGGT1837              GAATGTAAATAATGATAATAGCAATGATACATGTTGGAATCTCGTTTTGTTCTTTGTGTG1897              CATAGGCGCTTTGGGGGTGTATTTTTAGGCGTTAGACTTATTTTCAATTCGTGTATAATG1957              CGGTCAGTAAATGAATCATCAATTATTCAAATGCAATGCTGTATACGTGAAACTATTGGG2017              TTAAGACGCAGCTACTAGCTGACTGCTTGGTTACTTTCTGTGTACACCGCATGC2071                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 479 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetProArgThrSerLeuLeuThrLeuAlaCysAlaLeuAlaThrGly                              151015                                                                        AlaSerAlaPheSerTyrGlyAlaAlaIleProGlnSerThrGlnGlu                              202530                                                                        LysGlnPheSerGlnGluPheArgAspGlyTyrSerIleLeuLysHis                              354045                                                                        TyrGlyGlyAsnGlyProTyrSerGluArgValSerTyrGlyIleAla                              505560                                                                        ArgAspProProThrSerCysGluValAspGlnValIleMetValLys                              65707580                                                                      ArgHisGlyGluArgTyrProSerProSerAlaGlyLysAspIleGlu                              859095                                                                        GluAlaLeuAlaLysValTyrSerIleAsnThrThrGluTyrLysGly                              100105110                                                                     AspLeuAlaPheLeuAsnAspTrpThrTyrTyrValProAsnGluCys                              115120125                                                                     TyrTyrAsnAlaGluThrThrSerGlyProTyrAlaGlyLeuLeuAsp                              130135140                                                                     AlaTyrAsnHisGlyAsnAspTyrLysAlaArgTyrGlyHisLeuTrp                              145150155160                                                                  AsnGlyGluThrValValProPhePheSerSerGlyTyrGlyArgVal                              165170175                                                                     IleGluThrAlaArgLysPheGlyGluGlyPhePheGlyTyrAsnTyr                              180185190                                                                     SerThrAsnAlaAlaLeuAsnIleIleSerGluSerGluValMetGly                              195200205                                                                     AlaAspSerLeuThrProThrCysAspThrAspAsnAspGlnThrThr                              210215220                                                                     CysAspAsnLeuThrTyrGlnLeuProGlnPheLysValAlaAlaAla                              225230235240                                                                  ArgLeuAsnSerGlnAsnProGlyMetAsnLeuThrAlaSerAspVal                              245250255                                                                     TyrAsnLeuMetValMetAlaSerPheGluLeuAsnAlaArgProPhe                              260265270                                                                     SerAsnTrpIleAsnAlaPheThrGlnAspGluTrpValSerPheGly                              275280285                                                                     TyrValGluAspLeuAsnTyrTyrTyrCysAlaGlyProGlyAspLys                              290295300                                                                     AsnMetAlaAlaValGlyAlaValTyrAlaAsnAlaSerLeuThrLeu                              305310315320                                                                  LeuAsnGlnGlyProLysGluAlaGlySerLeuPhePheAsnPheAla                              325330335                                                                     HisAspThrAsnIleThrProIleLeuAlaAlaLeuGlyValLeuIle                              340345350                                                                     ProAsnGluAspLeuProLeuAspArgValAlaPheGlyAsnProTyr                              355360365                                                                     SerIleGlyAsnIleValProMetGlyGlyHisLeuThrIleGluArg                              370375380                                                                     LeuSerCysGlnAlaThrAlaLeuSerAspGluGlyThrTyrValArg                              385390395400                                                                  LeuValLeuAsnGluAlaValLeuProPheAsnAspCysThrSerGly                              405410415                                                                     ProGlyTyrSerCysProLeuAlaAsnTyrThrSerIleLeuAsnLys                              420425430                                                                     AsnLeuProAspTyrThrThrThrCysAsnValSerAlaSerTyrPro                              435440445                                                                     GlnTyrLeuSerPheTrpTrpAsnTyrAsnThrThrThrGluLeuAsn                              450455460                                                                     TyrArgSerSerProIleAlaCysGlnGluGlyAspAlaMetAsp                                 465470475                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgHisGlyXaaArgXaaPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuAlaValProAlaSerArgAsnGlnSerThrXaaAspThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuAlaValProAlaSerArgAsnGlnSerSerGlyAspThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuTyrValGluMetMetGlnAsnGlnAlaGluGlnThrProLeuVal                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetMetGlnCysGlnAlaGluGlnGluProLeuValArgValLeuVal                              151015                                                                        AsnAspArgXaa                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LysAspProArg                                                                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      LysAspProArgAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 4                                                               (C) OTHER INFORMATION: /NOTE= "Can be His or Leu"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TyrTyrGlyXaaGlyAlaGlyAsnProLeuGlyProThrGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TyrTyrGlyHisGlyAlaGlyAsnProLeuGlyProThrGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 5                                                               (C) OTHER INFORMATION: /note= "Can be Gln or Asn"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ThrGlyTyrValXaaTyrValGlnMetGln                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AlaGlnProGlyGlnAlaAlaProLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaGlnProGlyGlnSerSerProLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      LeuTyrValGluMetMetGlnCysGlnAlaGluGlnGluProLeuVal                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      PheIleGluGlyPheGlnSerAspLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TyrAlaPheLeuLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlyLeuSerPheAlaArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ValIleAlaSerGlyGluLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      PheTyrGlnArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AspSerPheValArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Val or Tyr"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 2                                                               (C) OTHER INFORMATION: /note= "Can be Leu or Glu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 3                                                               (C) OTHER INFORMATION: /note= "Can be Val or Ser"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 4                                                               (C) OTHER INFORMATION: /note= "Can be Asn or Leu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 5                                                               (C) OTHER INFORMATION: /note= "Can be Asp or Gln"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      XaaXaaXaaXaaXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TyrGluSerLeuThrArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      SerAlaAlaSerLeuAsnSer                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      LeuLysAspProArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TyrProThrGluSerLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Tyr or Asp"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 2                                                               (C) OTHER INFORMATION: /note= "Can be Phe or Pro"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 3                                                               (C) OTHER INFORMATION: /note= "Can be Asn or Ala"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 4                                                               (C) OTHER INFORMATION: /note= "Can be any amino acid"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      XaaXaaXaaXaaGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 3                                                               (C) OTHER INFORMATION: /note= "Can be Asn or Pro"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 4                                                               (C) OTHER INFORMATION: /note= "Can be Asp or Phe"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "Can be Asp or Ser"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 7                                                               (C) OTHER INFORMATION: /note= "Can be Gly or Leu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 8                                                               (C) OTHER INFORMATION: /note= "Can be Phe or Val"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LeuGluXaaXaaLeuXaaXaaXaaThrLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      LeuGluAsnAspLeuSerGlyValThrLeuThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TyrTyrGlyHisGlyAlaGlyAsnProLeuGlyProThrGlnGlyVal                              151015                                                                        GlyTyrAlaAsnGluLeuIleAla                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ValTyrPheAlaGlnValLeuSer                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      ValThrPheAlaGlnValLeuSer                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      PheIleGluGlyPheGlnSerThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AsxTyrLeuGlnSerLeuLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AspTyrLeuGlnSerLeuLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AsnIleGluProPheGlnValAsn                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ValLeuValAsnAspArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      LeuAlaValProAlaSerArgAsnGlnSerThrCysAspThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      PheSerTyrGlyAlaAlaIleProGlnSerThrGlnGluLysGlnPhe                              151015                                                                        SerGlnGluPheArgAspGly                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      PheSerSerGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ArgHisGlyXaaArgXaaPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      ArgHisGlyGluArgTyrProSerProSerAlaGlyLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      PheSerTyrGlyAlaAlaIleProGlnSerThrGlnGluLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AspIleGluGluAlaLeuAlaLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Ser or Ala"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 3                                                               (C) OTHER INFORMATION: /note= "Can be Glu or Pro"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      XaaIleXaaGlu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AlaArgTyrGlyHisLeuTrpAsnGlyGluThrValValProPhePhe                              151015                                                                        SerSerGly                                                                     (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Ser or Arg"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 2                                                               (C) OTHER INFORMATION: /note= "Can be Tyr or His"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 4                                                               (C) OTHER INFORMATION: /note= "Can be Gly or Glu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 5                                                               (C) OTHER INFORMATION: /note= "Can be Asn or Arg"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "Can be Gly or Tyr"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 8                                                               (C) OTHER INFORMATION: /note= "Can be Tyr or Ser"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 10                                                              (C) OTHER INFORMATION: /note= "Can be Glu or Ser"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      XaaXaaGlyXaaXaaXaaProXaaProXaaAlaGly                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TyrGlyGlyAsnGlyProTyr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 7                                                               (C) OTHER INFORMATION: /note= "Can be any amino acid"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GlnPheSerGlnGluPheXaaAspGlyTyrArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GlnPheSerGlnGluPheArgAspGlyTyr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Thr or His"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 7                                                               (C) OTHER INFORMATION: /note= "Can be any amino acid"                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 8                                                               (C) OTHER INFORMATION: /note= "Can be Tyr or Pro"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      XaaTyrGlyGlyAsnGlyXaaXaa                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      PheSerSerGlyTyrGlyArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      ValAlaPheGlyAsnProTyr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 1                                                               (C) OTHER INFORMATION: /note= "Can be Asp or Glu"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-Site                                                   (B) LOCATION: 7                                                               (C) OTHER INFORMATION: /note= "Can be Phe or Lys"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      XaaLeuAsnAlaIleLeuXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GlnLeuProGlnPheLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      ValSerTyrGlyIleAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      LeuTyrValGluMetMetGlnCysGlnAlaGluGln                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GCTTGGCATTGTACTAC17                                                           (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GCCTGACACTGTACTAC17                                                           (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:cDNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "N is Inosine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GGGTANCGCTCGCCGTG17                                                           (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "N is Inosine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GGATANCTTTCACCATG17                                                           (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "N is Inosine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GGGTANCGCTCGCCGTG17                                                           (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (C) OTHER INFORMATION: /note= "N is Inosine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GGGTANCGCTCCCCGTG17                                                           (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      CAACTGCCGCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      CAGCTACCACAGTTCAA17                                                           (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CAACTTCCTCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      CAACTCCCCCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      CAATTACCGCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      CAGTTGCCACAGTTCAA17                                                           (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      CAATTACCTCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      CAATTACCCCAATTTAA17                                                           (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      GlyThrArgAsnGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      ArgCysThrArgAlaCys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      CTACCCCTCTGCATCTAG18                                                          (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      GAATTCCGAGTCCGAGGTCATGGGCGCG28                                                (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      GAATTCCCGGGACCTACCCCTCTGCAT27                                                 (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      AGAAGAAATTTCTAGAACAGCAGCGATTGG30                                              (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      CGAGAGCACCTTCTCTAGATTTTGTCAAATGTACC35                                         (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      ACCCTCACCGAACTTGCGGGCCG23                                                     (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      ArgHisGlyXaaArgXaaPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      AlaGlnValLeuSerArgHisGlyAlaArgTyrProThrGluSerLys                              151015                                                                        GlyLys                                                                        (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      ValIleMetValLysArgHisGlyGluArgTyrProSerProSerAla                              151015                                                                        GlyLys                                                                        (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      ValValIleValSerArgHisGlyValArgAlaProThrLysAlaThr                              151015                                                                        GlnLeu                                                                        (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      ValHisThrLeuGlnArgHisGlySerArgAsnProThrGlyGlyAsn                              151015                                                                        AlaAla                                                                        (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      LeuGlnMetValGlyArgHisGlyGluArgTyrProThrValSerLeu                              151015                                                                        AlaLys                                                                        (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      LeuGlnMetLeuAlaArgHisGlyGluArgTyrProThrTyrSerLys                              151015                                                                        GlyAla                                                                        (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      ValThrLeuLeuTyrArgHisGlyAspArgSerProValLysAlaTyr                              151015                                                                        ProLys                                                                        (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      ValThrLeuValPheArgHisGlyAspArgGlyProIleGluThrPhe                              151015                                                                        ProAsn                                                                        (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      ValThrLeuLeuTyrArgHisGlyAspArgSerProValLysThrTyr                              151015                                                                        ProLys                                                                        (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      ValThrLeuValPheArgHisGlyAspArgSerProIleAspThrPhe                              151015                                                                        ProThr                                                                        (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      CTAGACACCTCAGCAATGTCGTTCCGATCTCTACTCGCCCTGAGCGGCCTCGTCTGCACA60                GGGTTGGCACTGGCAGTCCCCGCC84                                                    (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      TCGAGGCGGGGACTGCCAGTGCCAACCCTGTGCAGACGAGGCCGCTCAGGGCGAGTAGAG60                ATCGGAACGACATTGCTGAGGTGT84                                                    __________________________________________________________________________

What is claimed is:
 1. A transformed recombinant host cell transformedwith a first nucleic acid molecule comprising a nucleotide sequenceencoding a phytase and a second nucleic acid molecule comprising anucleotide sequence selected from the group consisting of(a) anucleotide sequence encoding a pH 2.5 acid phosphatase having the aminoacid sequence of amino acids 1-479 of SEQ ID No. 4; and (b) a nucleotidesequence encoding a pH 2.5 acid phosphatase, wherein said nucleotidesequence encoding said pH 2.5 acid phosphatase(i) hybridizes to thecomplement of the DNA sequence set forth in the coding region of SEQ IDNo. 3; and (ii) remains hybridized to the complement of the DNA sequenceset forth in the coding region of SEQ ID No. 3 when subjected to ahybridization solution at 68° C. for 2 hours, wherein the salt in saidhybridization solution is provided by 0.1×SSC, and wherein said hostcell secretes the active phytase encoded by said nucleotide sequenceencoding said phytase, wherein said host cell secretes the active pH 2.5acid phosphatase encoded by said nucleotide sequence encoding said pH2.5 acid phosphatase, and wherein the amount of said phytase and theamount of said phosphatase secreted by said host cell are secreted in apredetermined, desired ratio.
 2. The transformed recombinant host cellof claim 1, wherein said first nucleic acid molecule comprises anucleotide sequence encoding a phytase having the amino acid sequence ofamino acids 1-467 of SEQ ID No.
 2. 3. The transformed recombinant hostcell of claim 2, wherein said first nucleic acid molecule comprises thenucleotide sequence set forth in the coding region of SEQ ID No.
 1. 4.The transformed recombinant host cell of claim 1, wherein said secondnucleic acid molecule comprises a nucleotide sequence encoding a pH 2.5acid phosphatase having the amino acid sequence of amino acids 1-479 ofSEQ ID No.
 4. 5. The transformed recombinant host cell of claim 4,wherein said second nucleic acid molecule comprises the nucleotidesequence set forth in the coding region of SEQ ID No.
 3. 6. Thetransformed recombinant host cell of claim 1, wherein said secondnucleicacid molecule comprises a nucleotide sequence encoding a pH 2.5acid phosphatase, wherein said nucleotide sequence(i) hybridizes to thecomplement of the DNA sequence set forth in the coding region of SEQ IDNo. 3; and (ii) remains hybridized to the complement of the DNA sequenceset forth in the coding region of SEQ ID No. 3 when subjected to ahybridization solution at 68° C. for 2 hours, wherein the salt in saidhybridization solution is provided by 0.1×SSC.
 7. The transformedrecombinant host cell of claim 1, wherein said phytase secreted by saidcell has an enzyme activity per milliliter that is at least about 2-foldgreater than the phytase enzyme activity secreted by ALKO243 (ATCC#38854) or said phosphatase secreted by said cell has an enzyme activitythat is at least about 10-fold greater than the phosphatase enzymeactivity secreted by ALKO243 (ATCC #38854).
 8. The transformedrecombinant host cell of claim 1, wherein said phosphatase secreted bysaid cell has an enzyme activity per milliliter that is about 3-fold toabout 16-fold greater than the enzyme activity of said phytase secretedby said cell.
 9. The transformed recombinant host cell of claim 1,wherein said cell is selected from the group consisting of a bacteriumand a fungus.
 10. The transformed recombinant host cell of claim 9,wherein said bacterium is selected from the group consisting of E. coliand Bacillus.
 11. The transformed recombinant host cell of claim 9,wherein said fungus is a filamentous fungus.
 12. The transformedrecombinant host cell of claim 11, wherein said filamentous fungus isselected from the group consisting of Aspergillus, Trichoderma,Penicillium, Cephalosa and Rhizopus.
 13. The transformed recombinanthost cell of claim 12, wherein said filamentous fungus is Aspergillus.14. The transformed recombinant host cell of claim 13, wherein saidfilamentous fungus is Aspergillus niger.
 15. The transformed recombinanthost cell of claim 14, wherein said Aspergillus niger is a strainselected from the group consisting of GAX-11, GAX-12, GBE-14, GBH-134,GBH-157, GBJ-76, and GBJ-82.
 16. A transformed recombinant host celltransformed with a nucleic acid molecule comprising a first nucleotidesequence encoding a phytase and a second nucleotide sequence selectedfrom the group consisting of(a) a nucleotide sequence encoding a pH 2.5acid phosphatase having the amino acidsequence of amino acids 1-479 ofSEQ ID No. 4; and (b) a nucleotide sequence encoding a pH 2.5 acidphosphatase, wherein said nucleotide sequence encoding said pH 2.5 acidphosphatase(i) hybridizes to the complement of the DNA sequence setforth in the coding region of SEQ ID No. 3; and (ii) remains hybridizedto the complement of the DNA sequence set forth in the coding region ofSEQ ID No. 3 when subjected to a hybridization solution at 68° C. for 2hours, wherein the salt in said hybridization solution is provided by0.1×SSC, and wherein said host cell secretes the active phytase encodedby said first nucleotide sequence encoding said phytase, wherein saidhost cell secretes the active pH 2.5 acid phosphatase encoded by saidsecond nucleotide sequence encoding said pH 2.5 acid phosphatase, andwherein the amount of said phytase and the amount of said phosphatasesecreted by said host cell are secreted in a predetermined desiredratio.
 17. The transformed recombinant host cell of claim 16, whereinsaid first nucleotide sequence encodes a phytase having the amino acidsequence of amino acids 1-467 of SEQ ID No.
 2. 18. The transformedrecombinant host cell of claim 16, wherein said first nucleotidesequence is set forth in the coding region of SEQ ID No.
 1. 19. Thetransformed recombinant host cell of claim 16, wherein said secondnucleotide sequence encodes a pH 2.5 acid phosphatase having the aminoacid sequence of amino acids 1-479 of SEQ ID No.
 4. 20. The transformedrecombinant host cell of claim 19, wherein said second nucleotidesequence is set forth in the coding region of SEQ ID No.
 3. 21. Thetransformed recombinant host cell of claim 16, wherein said secondnucleotide sequence encodes a pH 2.5 acid phosphatase, wherein saidsecond nucleotide sequence encoding said pH 2.5 acid phosphatase(i)hybridizes to the complement of the DNA sequence set forth in the codingregion of SEQ ID No. 3; and (ii) remains hybridized to the complement ofthe DNA sequence set forth in the coding region of SEQ ID No. 3 whensubjected to a hybridization solution at 68° C. for 2 hours, wherein thesalt in said hybridization solution is provided by 0.1×SSC.
 22. Thetransformed recombinant host cell of claim 16, wherein said phytasesecreted by said cell has an enzyme activity per milliliter that is atleast about 2-fold greater than the phytase enzyme activity secreted byALKO243 (ATCC #38854) or said pliosphatase secreted by said cell has anenzyme activity that is at least about 10-fold greater than thephosphatase enzyme activity secreted by ALKO243 (ATCC #38854).
 23. Thetransformed recombinant host cell of claim 19, wherein said secretedphosphatase has an enzyme activity per milliliter that is about 3-foldto about 16-fold greater than the enzyme activity of said secretedphytase.
 24. The transformed recombinant host cell of claim 16, whereinsaid cell is selected from the group consisting of a bacterium and afungus.
 25. The transformed recombinant host cell of claim 24, whereinsaid bacterium is selected from the group consisting of E. coli andBacillus.
 26. The transformed recombinant host cell of claim 24, whereinsaid fungus is a filamentous fungus.
 27. The transformed recombinanthost cell of claim 26, wherein said filamentous fungus is selected fromthe group consisting of Aspergillus, Trichoderma, Bacillium,Cephalosporium, and Rhizopus.
 28. The transformed recombinant host cellof claim 27, wherein said filamentous fungus is Aspergillus.
 29. Thetransformed recombinant host cell of claim 28, wherein said filamentousfungus is Aspergillus niger.
 30. The transformed recombinant host cellof claim 29, wherein said Aspergillus niger is a strain selected fromthe group consisting of GBJ-9, GBJ-10, GBJ-13, GBJ-16, GBJ-26, GBJ-27,GBJ-28, GBJ-31, GBJ-35, GBJ-38, GBJ-40.
 31. An enzymatically activemixture comprising an enzymatically active phytase and the enzymaticallyactive pH 2.5 acid phosphatase encoded by the coding region of SEQ IDNo. 3, wherein the ratio of the enzyme activity of said pH 2.5 acidphosphatase to the enzyme activity of said phytase is from about 3:1 toabout 16:1.
 32. A feed comprising the mixture of claim
 31. 33. A methodof increasing the release of minerals from phytate complexes in ananimal diet, said method comprising feeding said animal the feed ofclaim
 32. 34. A method of increasing the release of phosphates fromphytate complexes in a plant material, said method comprising incubatingthe mixture of claim 31 with said plant material.
 35. An enzymaticallyactive mixture comprising an active phytase and the active pH 2.5 acidphosphatase encoded by a nucleotide sequence that(i) hybridizes to thecomplement of the DNA sequence set forth in the coding regions of SEQ IDNo. 3; and (ii) remains hybridized to the complement of the DNA sequenceset forth in the coding regions of SEQ ID No. 3 when subjected to ahybridization solution at 68° C. for 2 hours, wherein the salt in saidhybridization solution is provided by 0.1×SSC, and wherein the ratio ofthe enzyme activity of said pH 2.5 acid phosphatase to the enzymeactivity of said phytase is from about 3:1 to about 16:1.
 36. A feedcomprising the mixture of claim
 35. 37. A method of increasing therelease of minerals from phytate complexes in an animal diet, saidmethod comprising feeding said animal the feed of claim
 36. 38. A methodof increasing the release of phosphates from phytate complexes in aplant material, said method comprising incubating the mixture of claim35 with said plant material.